Methods for treating and preventing neutrophil-derived net toxicity and thrombosis

ABSTRACT

Embodiments of the technology described herein are based upon the discoveries that neutrophil extracellular traps (NETs) provide a stimulus for thrombus formation and that NETs are present in stored blood products. Accordingly, some embodiments relate to methods of treating and preventing toxicity of NETs and thrombosis caused by NETs. Additional embodiments are directed towards methods of treating stored blood products to prevent transfusion-related injuries.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional under 35 U.S.C. § 121 of co-pendingU.S. application Ser. No. 16/802,918 filed Feb. 27, 2020, which is adivisional under 35 U.S.C. § 121 of U.S. application Ser. No. 15/474,012filed Mar. 30, 2017 now U.S. Pat. No. 10,617,742 issued Apr. 14, 2020,which is a divisional under 35 U.S.C. § 121 of U.S. application Ser. No.14/119,499 filed Mar. 25, 2014 now U.S. Pat. No. 9,642,822 issued May 9,2017, which is a 35 U.S.C. § 371 National Phase Entry Application ofInternational Application No. PCT/US2012/039613 filed May 25, 2012,which designates the U.S., and which claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 61/490,877 filed May 27,2011, the contents of which are incorporated herein by reference intheir entirety.

FEDERAL FUNDING

This invention was made with federal funding under Grant Nos. P01HL056949, R01 HL041002, R01 HL095091, and R01 HL102101 awarded by theNational Institutes of Health and the National Heart Lung and BloodInstitute. The U.S. government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 21, 2013, isnamed 701039-066002-US_SL.txt and is 9,077 bytes in size.

TECHNICAL FIELD

The technology described herein relates to methods of treating andpreventing toxicity and thrombosis caused by leukocyte-derivedextracellular DNA traps, such as neutrophil extracellular traps (NETs),in the circulatory system, organs (e.g. lungs), tissues, or in bloodproducts.

BACKGROUND

During an infection, the body's innate immune system will be activated,bringing a number of non-specific defensive mechanisms (as opposed tothe specific responses of the adaptive immune system such as antibodies)to bear on the threat. Neutrophils are one of the cell types involved inthe innate immune response. They will actively attack a pathogen byproducing a respiratory burst; exposing the pathogenic cell to hydrogenperoxide, free radicals, and hypochlorite. Neutrophils are alsophagocytic, meaning that they will engulf and then degrade pathogeniccells or damaged host cells; essentially “eating” a unwanted cell inorder to destroy it. When neutrophils themselves die, they can releasefibers of DNA associated with histones and a number of antimicrobialproteins. These neutrophil extracellular traps (NETs) entangle and killbacterial pathogens.

SUMMARY

Described herein is the discovery that treatment of subjects with DNaseor anti-histone antibodies reduces the level of NETs in the bloodstreamand can reduce the incidence and severity of stroke, deep veinthrombosis, and pulmonary thromboembolism. Accordingly, provided hereinare methods for treating patients to prevent the accumulation of harmfullevels of NETs or degrade existing levels of NETs, thus providing novelmethods of treating a number of cardiovascular conditions.

Also described herein is the discovery of the presence of NETs in storedblood products. In addition, the inventors have determined thattreatment of stored blood products with DNase reduces the accumulationof NETs in the blood product. Accordingly, in certain embodiments,provided herein are methods and devices for treating accumulations ofNETs in both stored blood products and a patient's blood in order toavoid NET-induced cytotoxicity and thrombosis.

In certain embodiments, provided herein are methods of degrading NETsand/or preventing the formation of NETs in both stored blood productsand a patient with anti-NET compounds. In certain embodiments, ananti-NET compound is selected from the group consisting of: DNase,RNAse, a histone-degrading enzyme, an inhibitor of chromatindecondensation, an antibody against a component of a NET, or a PAD4inhibitor.

In certain embodiments the methods provided herein involve the use of atleast one anti-NET compound. In further embodiments, the method providedherein involves the use of two or more anti-NET compounds.

In certain embodiments, the method provided herein is directed to thetreatment of stored blood products and comprises contacting the bloodproducts with an effective amount of at least one anti-NET compound. Infurther embodiments, the blood product can be contacted with an anti-NETcompound at the time of collecting the blood, at any point duringstorage of the blood product, or at the time of transfusing the bloodproducts into a patient.

In certain embodiments the anti-NET compound is contained in acomposition comprising the anti-NET compound and a pharmaceuticallyacceptable carrier. In further embodiments the anti-NET compound can becontained in a composition comprising a pharmaceutically acceptablecarrier and another compound which would be of use in prolonging theshelf-life, efficacy, or safety of a blood product.

In certain embodiments, contacting a blood product with a singleadministration of an anti-NET compound decreases the concentration ofNETs in a blood product by at least 10%, e.g., by at least 20%, at least30%, at least 50%, at least 75%, at least 90%, at least 95%, at least99% or more as compared to the blood product prior to treatment with theanti-NET compound.

In certain embodiments, the anti-NET compound can be added to the bloodstorage product once it has been collected. In further embodiments, theanti-NET compound can be provided in a blood collection device, a bloodstorage device, or a blood delivery device. In further embodiments, theanti-NET compound can be administered to a patient when they receive atransfusion of blood products.

In certain embodiments, the blood products can be whole blood, red bloodcells, blood plasma, or platelets.

In certain embodiments, the method provided herein is directed toprevention of transfusion-related acute lung injury (TRALI) bycontacting blood products with an effective amount of an anti-NETcompound.

In certain embodiments, the technology described herein relates to adevice containing an effective amount of an anti-NET compound. Infurther embodiments, the device can be a blood collection device, bloodstorage device, or blood delivery device.

In certain embodiments, the blood storage device is capable of holdingat least 75 mLs of blood product, e.g. at least 75 mL of blood product,at least 100 mL of blood product, at least 200 mL of blood product, atleast 500 mL of blood product, at least 1000 mL of blood product, atleast 2000 mL of blood product or more.

In certain embodiments, the method provided herein is directed totreatment or prevention of TRALI by administering to a patient aneffective amount of anti-NET compound. In certain embodiments, theanti-NET compound is administered by inhalation.

In certain embodiments, the method provided herein is directed totreatment of acute lung injury by administering to a patient aneffective amount of anti-NET compound. In certain embodiments, the acutelung injury is caused by embolism, ischemia, hyperoxia, inflammation,sepsis, pancreatitis, oleic acid, acid aspiration, sepsis, oropharyngealaspiration, and/or exposure to ozone, polytetrafluoroethylene, nickelsulfate, and/or lipopolysaccharide. In certain embodiments, the anti-NETcompound is administered by inhalation.

In certain embodiments, the method provided herein is directed to thetreatment or prevention of cardiovascular conditions caused by NETs byadministering to a patient an effective dose of an anti-NET compound. Ina further embodiment the cardiovascular condition is stroke, ischemicreperfusion, myocardial infarction, inflammation, or thrombosis.

In certain embodiments, the effective dose of an anti-NET compound isadministered to a patient prophylactically when they are at risk for acardiovascular condition or exhibiting symptoms, indicators, or markersthat a cardiovascular condition is likely to occur. In certainembodiments, the patient is at risk for thrombosis, e.g. DVT.

In certain embodiments, the effective dose of an anti-NET compound isadministered to a patient repeatedly.

In certain embodiments the composition comprising at least one anti-NETcompound further comprises a pharmaceutically acceptable carrier. Infurther embodiments, the composition comprising at least one anti-NETcompound further comprises another compound which would be useful intreating or preventing a cardiovascular condition.

In certain embodiments, a single administration of an anti-NET compoundto a patient decreases the concentration of NETs in the patient'sbloodstream by at least 10%, e.g., by at least 20%, at least 30%, atleast 50%, at least 75%, at least 75%, at least 90%, at least 95%, atleast 99% or more as compared to the blood product prior to treatmentwith the anti-NET compound.

In one embodiment, a single administration of an anti-NET compounddecreases the level of an indicator, symptom, or marker of acardiovascular condition by at least 10%, e.g., by at least 20%, atleast 30%, at least 50%, at least 75%, at least 75%, at least 90%, atleast 95%, at least 99% or more as compared to the level of theindicator, symptom, or maker of a cardiovascular condition prior totreatment with the anti-NET compound.

In one embodiment, a single administration of an anti-NET compound to agroup of patients decreases the incidence or severity of sepsis, stroke,thrombosis, infarction, ischemia or death by at least 10%, e.g., by atleast 10%, by at least 20%, at least 30%, at least 50%, at least 75%, aat least 75%, at least 90%, at least 95%, at least 99% or more ascompared to the incidence of sepsis, stroke, or death in a group ofpatients not administered the anti-NET compound.

In certain embodiments, the method provided herein is directed toassessing a thrombotic condition in a patient comprising determining thelevel of NETs in a sample obtained from a patient, wherein an increasein the level of NETs as compared to a reference is indicative that athrombotic event has occurred or is likely to occur.

In certain embodiments, the method provided herein is directed toassessing the efficacy of the administration of an effective dose of atleast one anti-NET compound comprising determining the level of NETsobtained from a patient before and after treatment with the anti-NETcompound, wherein a reduction in the level of NETs following thetreatment with the anti-NET compound is indicative of efficacy.

The details of various embodiments of the invention are set forth in thedescription below. Other features, objects, and advantages of theinvention will be apparent from the description and the drawings, andfrom the claims.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D show electron micrographs and graphs demonstrating that NETsprovide a scaffold for platelet adhesion and aggregation and that DNasedegrades NETs and inhibits platelet adhesion. FIG. 1A is an electronmicrograph depicting platelets (Pts) attached to a fibrous meshwork ofNETs (scale bar, 1 μm). FIG. 1B is an electron micrograph depictingfilopods present on the platelets (Pt) attached to NETs, indicating thatthe platelets were activated (scale bar, 0.5 μm).

FIG. 1C shows the quantification of fluorescently-labelled NETs in thepresence (open circles) or absence of DNase (closed circles). The x-axisrepresents the time elapsed and the y-axis shows arbitrary units ofNETs. DNase was added to untreated samples after 10 min (arrow). FIG. 1Dshows the quantification of fluorescently-labelled platelets in thepresence (open circles) or absence of DNase (closed circles). The x-axisrepresents the time elapsed and the y-axis shows arbitrary units ofplatelets. DNase was added to untreated samples after 10 min (arrow).Data presented are representative of at least three independentexperiments and are shown as mean±SEM, n=3.

FIGS. 2A-2E shows graphs and a western blot demonstrating that heparindismantles NETs and prevents histone induced platelet aggregation. FIG.2A shows the quantification of fluoresecently-labelled NETs following 10minutes of perfusion with blood in the presence or absence of heparin.The x-axis shows the heparin content of the blood and the y-axis showsarbitrary units (A.U.) of NET-DNA. FIG. 2B shows the quantification offluorescently-labelled platelets following 10 minutes of perfusion ofNETs with blood containing fluorescently labeled platelets and with orwithout heparin added to the blood. The x-axis shows the heparin contentof the blood and the y-axis shows arbitrary units (A.U.) of platelets.For FIGS. 2A and 2B data are presented as mean±SEM, n=3; (Student's ttest; *P<0.05; **P<0.01). FIG. 2C shows the immunodetection of histoneH2B (arrow) in the supernatant of NETs treated with DNase (DN) orvarious concentrations of heparin. A second band (arrowhead) mayrepresent cross reactivity of the antibody or a proteolytic product.Data presented are representative of three independent experiments. FIG.2D is a graph of the degree of aggregation of platelets treated withthrombin (open circles), human recombinant histone H3 (solid circles),EDTA (solid squares), and heparin (solid triangles). The x-axis showsthe elapsed time and the y-axis represents the percentage of lighttransmission. FIG. 2E is a graph of the degree of aggregation ofplatelets 3 minutes after addition of various histones or throbmin(Thr). The x-axis shows the treatment and the y-axis depicts thepercentage of light transmission. (ANOVA; ***P<0.001 compared withhistone H1).

FIGS. 3A-3D show light microscopy, electron microscopy, and graphsdemonstrating that NETS provide a scaffold for red blood cell (RBC)-richthrombi. FIG. 3A shows a red thrombus (arrow) anchored on two strings(arrowheads) in a flow chamber coated with NETs after perfusion withblood (scale bar, 500 μm). FIG. 3B is an electron micrograph showingindividual RBCs attached to NETs (scale bar, 5 μm). FIG. 3C is a graphof the quantification of RBCs (as determined by hemoglobin content)attached to collagen or NETs after perfusion with our without DNase. Thex-axis indicates whether the perfusion chamber was coated with collagenor NETs, the y-axis indicates the quantity of attached RBC in arbitraryunits (A.U.) and the legend indicates that white bars representperfusion without DNase while black bars represent perfusion in thepresence of DNase. Data presented are representative of at least threeindependent experiments and presented as mean±SEM, n=3; (ANOVA;**P<0.01); n.s.=not significant. FIG. 3D is a graph of thequantification of fluorescently-labelled platelets attached to collagenor NETs after perfusion with our without DNase. The x-axis indicateswhether the perfusion chamber was coated with collagen or NETs, they-axis indicates the quantity of attached platelets in arbitrary units(A.U.) and the legend indicates that white bars represent perfusionwithout DNase while black bars represent perfusion in the presence ofDNase. Data presented are representative of at least three independentexperiments and presented as mean±SEM, n=3; (ANOVA; **P<0.01); n.s.=notsignificant.

FIG. 4 is a graph showing that markers of NETs are abundant duringbaboon deep vein thrombosis (DVT). The y-axis shows the quantity ofplasma DNA measured in individual baboons. Baboons #1 and #3 are shownas black circles and babon #2 is shown in white circles. On the x-axis,baseline (BL) levels indicate measurements prior to inducing DVT. Alsoshow are measurements at 6 hours (6 h), 2 days (2 d), and 6 days (6 d)after DVT induction. Bars represent the mean value of groups (n=3;Repeated measures ANOVA; **P<0.01 compared with BL).

FIGS. 5A-5B show graphs depicting that NETs are elevated in stored bloodproducts. FIG. 5A graphs the amount of DNA in leukocyte-reduced(Leuko-R) and non leukocyte-reduced (Non-R) blood. FIG. 5B graphs theamount of nucleosomes in Leuko-R and Non-R blood. The x-axis depicts thetype of blood; fresh blood (ctrl), leukocyte-reduced (Leuko-R), or nonleukocyte-reduced (Non-R). The y-axis of FIG. 5A shows the amount of DNAdetected while the x-axis of FIG. 5B depicts the quantity of nucleosomesdetected.

FIG. 6 shows a graph indicating that DNaseII decreased the concentrationof NETs in stored blood products. The x-axis shows the amount of timethe blood was stored (in days) while the y-axis depicts the amount ofnucleosomes detected. Blood treated with DNase is shown in black circlesand a solid line while untreated blood (vehicle) is shown in whitecircles with dashed lines.

FIGS. 7A-7D show graphs indicating that NETs are increased aftercerebral ischemia/reperfusion. FIG. 7A shows the level of nucleosomesmeasured before and after a sham treatment. The x-axis shows thetimepoint of measurement and the y-axis shows the level of nucleosomesin arbitrary units (A.U.). Each line represents an individual animal.FIG. 7B shows the level of nucleosomes measured before and after atreatment causing a stroke. The x-axis shows the timepoint ofmeasurement and the y-axis shows the level of nucleosomes in arbitraryunits (A.U.). Each line represents an individual animal. FIG. 7C showsthe level of DNA measured before and after a sham treatment. The x-axisshows the timepoint of measurement and the y-axis shows the level of DNAin arbitrary units (A.U.). Each line represents an individual animal.FIG. 7D shows the level of DNA measured before and after a treatmentcausing a stroke. The x-axis shows the timepoint of measurement and they-axis shows the level of DNA in arbitrary units (A.U.). Each linerepresents an individual animal.

FIGS. 8A-8C show graphs depicting the cerebral structure and bloodcounts of DNase-1^(−/−) mice. FIG. 8A shows the scoring of developmentof the posterior communicating arteries (PComAs). The x-axis shows thegenotype of the mice; wildtype (WT) or DNase-1 knock-outs(DNase-1^(−/−)). The y-axis shows the development score. FIG. 8B is agraph of the percentage of regional cerebral blood flow (rCBF) before(baseline), during (occlusion), and after (reperfusion) induction ofstroke. Timepoints of measurement are shown on the x-axis while they-axis shows the percentage of regional cerebral blood flow. Wildtype(WT) mice are represented by black boxes and solid lines while DNase-1knock-outs (DNase-1^(−/−)) mice are represented by white boxes anddashed lines. FIG. 8C shows the cell counts of blood from wildtype mice(WT; black bars) or DNase-1 knock-out mice (DNase-1^(−/−); white bars).The cell type is shown on the x-axis. The left-hand y-axis shows thecell counts for leukocytes and neutrophils, while the right-hand y-axisshows the cell counts for platelets.

FIGS. 9A-9B show graphs and scores indicating that DNase-1^(−/−) miceare more prone to ischemic stroke. FIG. 9A shows the size of theinfarction (y-axis) in wildtype mice (WT) or DNase-1 knock-out mice(DNase-1^(−/−)) (x-axis). FIG. 9B shows scores for neurological functiontests which measure the functional severity of injury caused by stroke.

FIGS. 10A-10B show graphs and scores indicating that infusion ofrhDNase-1 protects mice from ischemic stroke. FIG. 10A shows the size ofthe infarction (y-axis) in control mice (vehicle) and mice treated withrhDNase-1 (rhDNase) (x-axis). FIG. 10B shows scores for neurologicalfunction tests which measure the functional severity of injury caused bystroke.

FIG. 11 shows the characterization of the BWA3 antibody's binding todifferent histones. The x-axis lists different histones or a mixedpopulation of histones (histones). The y-axis listing the degree ofantibody binding in arbitrary units (A.U.). BWA3 binding is shown inblack bars while the control IgG1 binding is shown in white bars.

FIGS. 12A-12B show graphs and scores indicating that histoneneutralization is protective during ischemic stroke. FIG. 12A shows thesize of the infarction (y-axis) in control mice (vehicle), mice treatedwith BWA3 histone-binding antibody (BWA3), and mice treated with controlIgG1 (IgG1) (x-axis). FIG. 12B shows scores for neurological functiontests which measure the functional severity of injury caused by stroke.

FIGS. 13A-13F show graphs indicating DNase 1 infusion protects mice fromflow restriction-induced thrombosis. Wild-type mice underwent IVCstenosis for 6 h (13A-13C) or 48 h (13D-13F). Mice received infusion ofeither vehicle or DNase 1 (50 l.lg i.p. and 10 l.tg i.v.) before thesurgery (13A-13F) and every 12 h thereafter (13D-13F). (13A, 13D)Thrombus weight and (13B, 13E) thrombus length are presented; horizontallines represent median. FIGS. 13C and 13F depict the percentage of micewith a thrombus. 6 h vehicle, n=14; 6 h DNase 1, n=10; 48 h vehicle.n=8; 48 h DNase 1, n=12.

FIG. 14 shows a graph indicating that DNase protects mice from pulmonaryembolism. The x-axis indicates whether mice were treated with controlsolutions (buffer), or administered the anti-NET compounds DNaseI orDNaseII. The number of mice in each group (n) is also specified. They-axis shows the % morality for each group following chemical inductionof pulmonary embolism.

FIGS. 15A-15C show scanning electron microscopy images illustrating theeffect of DNase treatments on TRALI-associated DNA-fibrous networks atthe alveoli surface. Scale bar is 1 μm, insets show low magnificationviews where scale bar is 2 μm. Alveolar sacs (a) and ducts (d) aremarked. FIG. 15A shows the lung epithelium of a normal, healthy mouselung. FIG. 15B shows the lung epithelium of a mouse in which TRALI hasbeen induced. FIG. 15C shows the lung epithelium of a mouse in whichTRALI has been induced and DNase has been administered intranasally.

FIGS. 16A-16B show graphs indicating that DNaseI treatment is protectiveduring TRALI. DNaseI or a vehicle control was administered intranasally10 min prior to anti-H2K^(d) antibody administration. FIG. 16A showsplatelet counts and FIG. 16B shows the protein concentration found inbronchial alveolar lavage (BAL).

FIGS. 17A-17B show graphs that depict NETosis assays in PAD4-deficientmice and their littermates. Neutrophils were isolated from peripheralblood and stimulated with either LPS from Klebsiella pneumoniae (10 μM,grey bars), calcium ionophore (4 μM ionomycin, black bars), or vehiclecontrol (RPMI+10 mM HEPES, white bars). After two hours, cells werefixed with 2% paraformaldehyde overnight and immunostaining wasperformed using anti-histone H3 (citrulline 2,8,17) antibody paired withALEXA FLUOR® 488-conjugated goat anti-rabbit IgG. DNA was counterstainedwith Hoeschst 33342. Fluorescent images were acquired using a ZeissAXIOVERT™ 200 inverted widefield fluorescence microscope in conjunctionwith a Zeiss AXIOCAM MRM™ monochromatic CCD camera and Zeiss AXIOVISION™software. Images were quantified using IMAGEJ™ software. The x-axesindicate whether the cells were isolated from wild-type mice (PAD4+/+),PAD4-deficient mice (PAD4−/−) or mice heterozygous for PAD4 (PAD4+/−).FIG. 17A shows a graph depicting quantification of H3Cit+ cells inisolated mouse neutrophils stimulated in vitro. FIG. 17B shows a graphdepicting quantification of NETs in isolated mouse neutrophilsstimulated in vitro. An unpaired Student's t test was performed todetermine significance. Error bars represent mean+/−s.e.m., * p<0.05.PAD4±/±mice, n=4, PAD4+/− mice, n=3, PAD4−/− mice, n=4.

FIGS. 18A and 18B show graphs depicting decreased thrombus size andfrequency of formation in PAD4−/− mice compared to wild-type C57Bl/6Jmice from 48 h DVT in the venous stenosis model. The inferior vena cavawas surgically exposed 48 h after ligation and the IVC opened betweenthe renal and iliac veins to expose thrombi for harvest. FIG. 18A showsa graph that depicts length of thrombi harvested from 48 h DVT in thevenous stenosis model. An unpaired Student's t test was performed todetermine significance. FIG. 18B shows a graph that depicts thepercentage of thrombi formed in each group. A contingency table andchi-square test was performed to determine significance. Error barsrepresent medians, *** p<0.001. Wild-type C57Bl/6J mice, n=10, PAD4−/−mice, n=11. Mice deficient in PAD4 do not form DVT.

FIGS. 19A-19C depict graphs of the binding of von Willebrand factor(VWF), fibronectin, and fibrinogen to neutrophil extracellular traps(NETs). Human NETs were incubated with human plasma for 30 min, washed,and stained for VWF (19A), fibronectin (19B), or fibrinogen (19C).Quantification showed that all three proteins were binding to NETs in aDNase-sensitive manner. Data are presented as mean±SEM, n=3; (Student'st test); **P<0.01; ***P<0.001.

FIG. 20 depicts a graph demonstrating that NETs intercalate within afibrin clot and generate a tissue plasminogen activator-(tPA) resistantscaffold. Fibrin polymerization was induced in blood by recalcificationand incubated either in the absence (medium) or presence of neutrophilspreactivated by PAF to induce NET formation (neutrophils+PAF). Sampleswere treated with DNase to digest extracellular traps or tPA for fibrindigestion. In the absence of activated neutrophils, clots were rich inRBCs, coincubation with DNase had no effect, and tPA alone, or togetherwith DNase prevented clot formation. In the presence of activatedneutrophils, blood clot appearance and size did not change even in thepresence of DNase. The tPA reduced clot size and prevented clotformation only in combination with DNase. (Scale bar, 1 cm.) The graphpresents quantification of the clots by weighing. The presence of PAF orunstimulated neutrophils alone did not produce a tPA-resistant clot.Data are presented as mean±SEM, n=4; (ANOVA; ***P<0.001). The first barin each x-axis category represents the untreated samples, the second barrepresents the samples treated with DNase, the third bar represents thesamples treated with tPA, and the fourth bar represents the samplestreated with tPA and DNase.

FIG. 21 depicts a graph demonstrating that plasma DNA level is elevated6 h after IVC stenosis application. Blood was drawn from wild-type micebefore and 6, 24 and/or 48 h after IVC stenosis application (DVT) orsham surgery. Plasma was prepared and DNA levels determined bySYTOXGREEN™ dye; n=3-8 per time point. *P<0.003 vs. the same mice atbaseline (paired t-test). #P<0.03, 6 h DVT vs. sham-operated mice(Mann-Whitney test). Error bars represent SEM.

FIGS. 22A-22C depict graphs demonstrating that histone infusion promotesflow restriction-induced thrombosis in mice. Histone mix (10 mg kg⁻¹)was infused into WT mice immediately before DVT surgery. Mice weresacrificed after 1 h stenosis and thrombi developed in the IVC wereexamined and harvested. Values for weight (FIG. 22A) and length (FIG.22B) of the thrombi are shown with medians (horizontal bars). (FIG. 22C)Percentage of mice that developed a thrombus. Vehicle-treated mice,n=14; histone-treated mice, n=9.

FIGS. 23A-23C demonstrate the effect of anti-HNA-3a antibody on NETformation by human neutrophils. FIG. 23A depicts a graph of thequantification of NETs following anti-HNA-3a antibody treatment byfluorescence microscopy analysis. HNA-3a-positive neutrophils primedwith TNF-α were incubated for 180 minutes with PBS (TNF-α+PBS), controlIgG purified from healthy volunteer plasma (TNF-α+Ctrl Ab), anti-HNA-3aantibody purified from two donors whose plasma induced TRALI (TNF-α+Ab1and TNF-α+Ab2) or 25 nM PMA as a positive control (TNF-α+PMA). DNArelease was visualized after DNA staining with Hoechst 33342. Theexperiment was independently performed nine times. Bars representmeans±s.e.m. FIG. 23B depicts examples of the fluorescence imagesquantified in (FIG. 23A). White arrowheads point at selected cellsforming NETs. White arrows point at examples of delobulated (large)neutrophil nuclei. Their presence indicates nuclear decondensation, anearly step in NETosis. Scale bar, 50 FIG. 23C depicts a graph of thequantification of NETs following anti-HNA-3a F(ab′)₂ fragment treatmentby fluorescence microscopy analysis. HNA-3a-positive neutrophils wereincubated and analyzed in the same conditions as in (FIG. 23A) but werealso treated with anti-HNA-3a F(ab′)₂ fragments (TNF-α+F(ab′)₂anti-HNA-3a). The experiment was independently performed three times.Bars represent means±s.e.m.

FIG. 24 depicts a graph demonstrating that inhibition of anti-HNA-3aantibody-induced NETs formation by FcγRIIa blockade. Isolatedneutrophils were treated for 15 minutes with PBS or with a CD32(FcγRIIa)-blocking antibody, primed with TNF-α and incubated for 180minutes with control IgG purified from healthy volunteer blood(TNF-α+Ctrl Ab) or with anti-HNA-3a antibodies purified from two blooddonors whose blood/blood product induced TRALI (TNF-α+Ab1 andTNF-α+Ab2). DNA release was visualized after DNA staining with Hoechst33342 and fixation. The experiment was independently performed 3 times.Bars represent means±s.e.m. An overall increase in background isobserved. This is likely due to the additional incubation of neutrophilsduring PBS or anti-CD32 antibody pretreatment. *** P<0.005.

FIGS. 25A-25D depict graphs characterizing the TRALI model in BALB/cmice used in this study. (25A) Peripheral neutrophil and (25B) plateletcounts, (25C) BAL proteins and (25D) lung tissue MPO content in miceadministered i.p. with LPS (0.1 mg/kg) and i.v. with an isotype controlor the anti-H-2Kd Ab (1.0 mg/kg, LPS group and TRALI group) representedby box plot. *P<0.05; **P<0.01.

FIGS. 26A-26C depict graphs providing evidence of circulating NETbiomarkers in the mouse model of TRALI. Quantification of (FIG. 26A)DNA, (FIG. 26B) nucleosomes and (FIG. 26C) myeloperoxidaseconcentrations in plasma from a group of mice challenged i.p. with LPS(0.1 mg/kg) and i.v. with an isotype control antibody (1 mg/kg)(LPS+Ctrl Ab) (n=7, 7 and 4 for FIGS. 26A, 26B and 26C respectively),and from a TRALI group that received both LPS and the anti-H-2K^(d)antibody (1 mg/kg) (LPS+H-2K^(d) Ab) (n=7, 7 and 4 for FIGS. 26A, 26Band 26C respectively). Blood was taken 2 hours after antibodyinjection. * P<0.05.

FIGS. 27A-27D depict electron micrographs demonstrating that a DNA-basedfibrous mesh is present in the lungs of mice with TRALI. Representativephotographs obtained by transmission electron microscopy of the lungepithelial surface of control mice (FIG. 27A), mice with TRALI (FIGS.27B, 27D) and mice that were injected with LPS (0.5 mg/kg) and receivedDNase 1 prior to anti-H-2K^(d) mAb injection (FIG. 27C). The fibrousmaterial found in TRALI lungs is absent after DNase 1 treatment. Scalebar, 1 μm. The insets show low magnification views of the lung surfacerevealing alveolar sacs (a) and ducts (d); inset scale bars, 2 μm. Thepictures are representative of three independent experiments.

FIGS. 28A-28C depict graphs demonstrating that DNase 1 treatmentimproves blood oxygenation of mice with TRALI. The mice received i.n.vehicle-buffer or i.n. DNase 1 10 minutes prior to anti-H-2K^(d)antibody injection. Mice that received DNase 1 showed more stablearterial blood oxygen saturation and improved transient hypoxia whencompared to the mice pretreated with the vehicle-buffer. (FIGS. 28A,28B) Mean (FIG. 28A) and minimum (FIG. 28B) of the arterial oxygensaturation recorded for 20 minutes 2 hours after antibody infusion incontrol mice (n=17), mice with TRALI (n=9), mice with TRALI that havereceived DNase 1 as a pretreatment 10 minutes before antibody infusion(n=7; Pre) or as a treatment 90 minutes after antibody infusion (n=9;Post). Mice with TRALI received i.n. either the vehicle-buffer or DNase1 (50 μg/mouse). Bars represent medians. * P<0.05; ** P<0.01. (FIG. 28C)Shock-like condition was determined by rectal temperature measurement inthe mice monitored for arterial oxygen saturation and additional controlmice (control group: n=25). Bars represent medians. ** P<0.01; ***P<0.005.

DETAILED DESCRIPTION Definitions

For convenience, the meaning of certain terms and phrases used in thespecification, examples, and appended claims, are provided below. Ifthere is an apparent discrepancy between the usage of a term in the artand its definition provided herein, the definition provided within thespecification shall prevail.

Definitions of common terms in cell biology and molecular biology can befound in “The Merck Manual of Diagnosis and Therapy”, 18th Edition,published by Merck Research Laboratories, 2006 (ISBN 0-911910-18-2);Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology,published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); andRobert A. Meyers (ed.), Molecular Biology and Biotechnology: aComprehensive Desk Reference, published by VCH Publishers, Inc., 1995(ISBN 1-56081-569-8); The ELISA guidebook (Methods in molecular biology149) by Crowther J. R. (2000); Fundamentals of RIA and Other LigandAssays by Jeffrey Travis, 1979, Scientific Newsletters; Immunology byWerner Luttmann, published by Elsevier, 2006. Definitions of commonterms in molecular biology are also be found in Benjamin Lewin, GenesIX, published by Jones & Bartlett Publishing, 2007 (ISBN-13:9780763740634); Kendrew et al. (eds.), Molecular Biology andBiotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8) and Current Protocols inProtein Sciences 2009, Wiley Intersciences, Coligan et al., eds.

Unless otherwise stated, the present invention was performed usingstandard procedures, as described, for example in Methods in Enzymology,Volume 289: Solid-Phase Peptide Synthesis, J. N. Abelson, M. I. Simon,G. B. Fields (Editors), Academic Press; 1st edition (1997) (ISBN-13:978-0121821906); U.S. Pat. Nos. 4,965,343, and 5,849,954; Maniatis etal., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., USA (1982); Sambrook et al.,Molecular Cloning: A Laboratory Manual (2 ed.), Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., USA (1989); Davis et al.,Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc.,New York, USA (1986); or Methods in Enzymology: Guide to MolecularCloning Techniques Vol. 152, S. L. Berger and A. R. Kimmerl Eds.,Academic Press Inc., San Diego, USA (1987); Current Protocols in ProteinScience (CPPS) (John E. Coligan, et. al., ed., John Wiley and Sons,Inc.), Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et.al. ed., John Wiley and Sons, Inc.), and Culture of Animal Cells: AManual of Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5thedition (2005), Animal Cell Culture Methods (Methods in Cell Biology,Vol. 57, Jennie P. Mather and David Barnes editors, Academic Press, 1stedition, 1998) which are all incorporated by reference herein in theirentireties.

The terms “decrease”, “reduced”, “reduction”, “decrease” or “inhibit”are all used herein generally to mean a decrease by a statisticallysignificant amount. However, for avoidance of doubt, “reduced”,“reduction” or “decrease” or “inhibit” means a decrease by at least 10%as compared to a reference level, for example a decrease by at leastabout 20%, or at least about 30%, or at least about 40%, or at leastabout 50%, or at least about 60%, or at least about 70%, or at leastabout 80%, or at least about 90%. In some embodiments, the decrease isup to and including a 100% decrease (e.g. absent level or non-detectablelevel as compared to a reference sample), or any decrease between10-100% as compared to a reference level.

The terms “increased”, “increase” or “enhance” or “activate” are allused herein to generally mean an increase by a statically significantamount; for the avoidance of any doubt, the terms “increased”,“increase” or “enhance” or “activate” means an increase of at least 10%as compared to a reference level, for example an increase of at leastabout 20%, or at least about 30%, or at least about 40%, or at leastabout 50%, or at least about 60%, or at least about 70%, or at leastabout 80%, or at least about 90% or up to and including a 100% increaseor any increase between 10-100% as compared to a reference level, or atleast about a 2-fold, or at least about a 3-fold, or at least about a4-fold, or at least about a 5-fold or at least about a 10-fold increase,or any increase between 2-fold and 10-fold or greater as compared to areference level.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means a two standard deviation(2SD) below normal, or lower, concentration of the marker. The termrefers to statistical evidence that there is a difference. It is definedas the probability of making a decision to reject the null hypothesiswhen the null hypothesis is actually true. The decision is often madeusing the p-value.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages canmean±1%.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of thisdisclosure, suitable methods and materials are described below. Theabbreviation, “e.g.” is derived from the Latin exempli gratia, and isused herein to indicate a non-limiting example. Thus, the abbreviation“e.g.” is synonymous with the term “for example.”

All patents and other publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that might beused in connection with the present invention. These publications areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing in this regard should be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention or for any other reason. Allstatements as to the date or representation as to the contents of thesedocuments is based on the information available to the applicants anddoes not constitute any admission as to the correctness of the dates orcontents of these documents.

As used herein, the phrase “contacting a blood product” refers to addingat least one anti-NET compound to a blood product or adding a bloodproduct to at least one anti-NET compound such that the blood productand anti-NET compound can interact with one another.

As used herein, the term “administer” refers to the placement of acomposition into a subject by a method or route which results in atleast partial localization of the composition at a desired site suchthat desired effect is produced. A compound or composition describedherein can be administered by any appropriate route known in the artincluding, but not limited to, oral or parenteral routes, includingintravenous, intramuscular, subcutaneous, transdermal, airway (aerosol),pulmonary, nasal, rectal, and topical (including buccal and sublingual)administration.

Exemplary modes of administration include, but are not limited to,injection, infusion, instillation, inhalation, or ingestion. “Injection”includes, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intraventricular, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal,intracerebro spinal, and intrasternal injection and infusion. Inpreferred embodiments, the compositions are administered by intravenousinfusion or injection.

As used herein, the term “antibody” refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site that immunospecificallybind an antigen. The terms also refers to antibodies comprised of twoimmunoglobulin heavy chains and two immunoglobulin light chains as wellas a variety of forms besides antibodies; including, for example, Fv,Fab, and F(ab)′2 as well as bifunctional hybrid antibodies (e.g.,Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)) and single chains(e.g., Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85, 5879-5883(1988) and Bird et al., Science 242, 423-426 (1988), which areincorporated herein by reference). (See, generally, Hood et al.,Immunology, Benjamin, N.Y., 2ND ed. (1984), Harlow and Lane, Antibodies.A Laboratory Manual, Cold Spring Harbor Laboratory (1988) andHunkapiller and Hood, Nature, 323, 15-16 (1986), which are incorporatedherein by reference).

As used herein in the context of expression, the terms “treat,”“treatment,” and the like, refer to an decrease in the concentration ofNETs in blood products or the bloodstream or decrease in the severity orincidence of cardiovascular conditions as described herein. In thecontext of the present invention insofar as it relates to any of theconditions recited herein, the terms “treat,” “treatment,” and the likemean to relieve, alleviate, ameliorate, inhibit, slow down, reverse, orstop the progression, aggravation, deterioration, progression,anticipated progression or severity of at least one symptom orcomplication associated with such condition. In one embodiment, thesymptoms of a condition are alleviated by at least 5%, at least 10%, atleast 20%, at least 30%, at least 40%, or at least 50%.

By “lower” in the context of a disease marker or symptom is meant astatistically significant decrease in such level. The decrease can be,for example, at least 10%, at least 20%, at least 30%, at least 40% ormore, and is preferably down to a level accepted as within the range ofnormal for an individual without such disorder.

As used herein, the phrase “effective amount” as used in relation toanti-NET compounds in a blood product refers to the amount of anti-NETcompound that provides a statistically significant decrease in theconcentration of NETs in the blood product as compared to theconcentration of NETs in the absence of the compound.

As used herein, the phrase “therapeutically effective amount” or“effective dose” refers to an amount that provides a therapeutic benefitin the treatment, prevention, or management of a cardiovascularcondition or a condition caused by NETs in blood products, e.g. anamount that provides a statistically significant decrease in at leastone symptom of a cardiovascular condition. Determination of atherapeutically effective amount is well within the capability of thoseskilled in the art. Generally, a therapeutically effective amount canvary with the subject's history, age, condition, sex, as well as theseverity and type of the medical condition in the subject, andadministration of other pharmaceutically active agents.

As used herein, the term “pharmaceutical composition” refers to theactive agent in combination with a pharmaceutically acceptable carrierof chemicals and compounds commonly used in the pharmaceutical industry.The term “pharmaceutically acceptable carrier” excludes tissue culturemedium.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject agents fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation, for example the carrierdoes not decrease the impact of the agent on the treatment. In otherwords, a carrier is pharmaceutically inert.

As used herein, a “subject” means a human or animal. In one embodiment,the animal is a vertebrate such as a primate, rodent, domestic animal,avian species, fish or game animal. The terms, “patient”, “individual”and “subject” are used interchangeably herein.

Preferably, the subject is a mammal. The mammal can be a human,non-human primate, mouse, rat, dog, cat, horse, or cow, but are notlimited to these examples. Mammals other than humans can beadvantageously used as subjects that represent animal models ofcardiovascular conditions. In addition, the methods described herein canbe used to treat domesticated animals and/or pets. A subject can be maleor female. A subject can be one who has been previously diagnosed with acardiovascular condition, or a subject identified as having one or morecomplications related to a cardiovascular condition, and optionally, butneed not have already undergone treatment for the cardiovascularcondition or the one or more complications related to the cardiovascularcondition. A subject can also be one who is not suffering from acardiovascular condition. For example, a subject can be one who exhibitsone or more risk factors for a cardiovascular condition, or one or morecomplications related to a cardiovascular condition. A subject can beasymptomatic for a cardiovascular condition or one or more complicationsrelated to a cardiovascular condition. In one embodiment, the subject isselected for having, or being at risk for having, a cardiovascularcondition. A subject can also be one who has been diagnosed with oridentified as having one or more complications related to acardiovascular condition, or alternatively, a subject can be one who hasnot been previously diagnosed with or identified as having one or morecomplications related to a cardiovascular condition.

NETs

Embodiments of the technology described herein are based on thediscovery that NETs provide a previously unrecognized scaffold andstimulus for thrombus formation. Upon activation, neutrophils and othercells undergo a cell death program termed “NETosis” (Fuchs et al., JCB2007 176:231-241) and release portions of nuclear DNA in the form ofnucleosomes in complex with various proteins having antimicrobialactivity (Brinkmannn et al. Science 2004 303:1532-5). These complexesare termed NETs and they are capable of entangling microbes and resultin degradation of virulence factors and killing of microbes (Brinkmannet al. Science 2004 303:1532-5). Release of NETs from neutrophils hasbeen associated with inflammation during sepsis and noninfectiousdiseases (Brinkmann et al. Science 2004 303:1532-5; Clark et al. NatureMedicine 2007 13:463-9; Gupta et al., Hum Immnol 2005 66:1146-1154;Kessenbrock et al. Nature Medicine 2009 15:623-5).

As used herein, the term “NET” refers to extracellular complexes ofnucleosomes and proteins, e.g. proteins having anti-microbial activity.The nucleosomes may be derived from neutrophils, mast cells,eosinophils, monocytes, or leukocytes.

The results described herein and by the inventors in Fuchs et al. PNAS2010 107:15880-15885 indicate that NETs are a previously unrecognizedlink between inflammation and thrombosis. Described herein is thediscovery that the histone component of NETs encourages thrombosis bypromoting platelet adhesion, activation, and aggregation and datademonstrating that NETs recruit red blood cells, promote fibrindeposition, and induce red thrombus, a marker of venous thrombosis.Herein, it has been further demonstrated that presence of NETS inthrombosis and plasma in a baboon model of deep vein thrombosis. Inaddition, the studies described herein have demonstrated that DNase orheparin treatment disassembles NETs and abrogates their thromboticstimulation properties. Accordingly, as described herein, thromboticconditions can be treated or prevented by the disruption of NETs.

Anti-NET Compounds

Some embodiments are directed to methods and devices for the treatmentof stored blood products with anti-NET compounds. Other embodiments aredirected to methods for the treatment or prevention of a cardiovascularcondition in a patient with anti-NET compounds. As used herein,“anti-NET compounds” can include any compound that degrades or targetsfor degradation any component of a NET and/or prevents the formation ofNETs (e.g. PAD4 inhibitors). Also included are compounds that otherwiseinhibit the activity of a NET component or impair the ability of a cellto form a NET, e.g. inhibition of PAD4, which is required for NETformation as described herein. An anti-NET compound can be a nucleicacid (DNA or RNA), small molecule, lipid, carbohydrate, protein,peptide, antibody, or antibody fragment. In some embodiments, ananti-NET compound can be an enzyme, e.g. an enzyme which cleaves and/ordegrades, e.g. a nucleic acid, protein, polypeptide, or carbohydrate. Asused herein, the term “small molecule” refers to a chemical agent whichcan include, but is not limited to, a peptide, a peptidomimetic, anamino acid, an amino acid analog, a polynucleotide, a polynucleotideanalog, an aptamer, a nucleotide, a nucleotide analog, an organic orinorganic compound (i.e., including heteroorganic and organometalliccompounds) having a molecular weight less than about 10,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 5,000 grams per mole, organic or inorganic compounds having amolecular weight less than about 1,000 grams per mole, organic orinorganic compounds having a molecular weight less than about 500 gramsper mole, and salts, esters, and other pharmaceutically acceptable formsof such compounds.

In certain embodiments an anti-NET compound can be, but is not limitedto; DNase, RNase, heparin, an antibody (i.e. an antibody to histones orto a particular histone), a histone degrading enzyme (i.e. mast cellproteinase 1 (Gene ID:1215)), plasmin (Gene ID: 5340), cathepsin D (GeneID:1509) or activated protein C (Gene ID:5624)) or an inhibitor ofchromatin decondensation (i.e. staurosporine, HDAC inhibitors (i.e.M344), PAD4 inhibitors, or elastase inhibitors (i.e. Gelin®)).

In one embodiment, the anti-NET compound is not heparin. In oneembodiment, the anti-NET compound is not RNase. In some embodiments, theanti-NET compound is selected from the group consisting of DNase; ahistone-degrading enzyme; an inhibitor of chromatin decondensation; anantibody against a component of a NET; an elastase inhibitor; or a PAD4inhibitor.

Anti-NET compounds can be produced recombinantly using methods wellknown to those of skill in the art (see Sambrook et al., MolecularCloning: A Laboratory Manual (2 ed.), Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., USA (1989)). Alternatively, anti-NETcompounds are available commercially e.g. Pulmozyme® (Genentech; SanFrancisco, Calif.), DNase (#D5319 Sigma-Aldrich; St. Louis, M0)(#90083Thermo Scientific; Rockford, Ill.), RNAse (#R4642 Sigma-Aldrich; St.Louis, Mo.), Heparin® (Celsus; Cincinatti, Ohio), anti-histoneantibodies (ab1791, ab8580, ab8898, ab6002, ab1790, ab9053, ab10158,ab71594, ab4269 Abcam; Cambridge, Mass.), mast cell proteinase 1(5146-SE-010 R&D Systems; Minneapolis, Minn.), thrombin (HCT-0020Haematologic Technologies; Essex Junction, Vt.), plasmin (HCPM-0140Haematologic Technologies; Essex Junction, Vt.), cathepsin D(1014-AS-010 R&D Systems; Minneapolis, Minn.), activated protein C(AEZ004B Aniara; Mason, Ohio), staurosporine (S4400 Sigma-Aldrich; St.Louis, Mo.), M344 (M5820 Sigma-Aldrich; St. Louis, Mo.) or Gelin® (G0528Sigma-Aldrich; St. Louis, Mo.).

In certain embodiments, the anti-NET compound is a monoclonal antibody(See, generally, Hood et al., Immunology, Benjamin, N.Y., 2ND ed.(1984), Harlow and Lane, Antibodies. A Laboratory Manual, Cold SpringHarbor Laboratory (1988) and Hunkapiller and Hood, Nature, 323, 15-16(1986), which are incorporated herein by reference). Monoclonalantibodies are prepared using methods well known to those of skill inthe art. Typically, spleen cells from an animal immunized with a desiredantigen are immortalized, commonly by fusion with a myeloma cell (see,Kohler and Milstein (1976) Eur, J. Immunol. 6:511-519, incorporatedherein by reference). Alternative methods of immortalization includetransformation with Epstein Barr Virus, oncogenes, or retroviruses, orother methods known in the art. Colonies arising from singleimmortalized cells are screened for production of antibodies of thedesired specificity and affinity for the antigen, and yield of themonoclonal antibodies produced by such cells may be enhanced by varioustechniques, including injection into the peritoneal cavity of avertebrate host. Alternatively, one may isolate DNA sequences whichencode a monoclonal antibody or a binding fragment thereof by screeninga DNA library from human B cells according, e.g., to the generalprotocol outlined by Huse, et al. (1989) Science 246:1275-1281.

Antibodies, including binding fragments and single chain versions,against predetermined fragments of a NET can be raised by immunizationof animals with conjugates of the fragments with carrier proteins.Monoclonal antibodies are prepared from cells secreting the desiredantibody. These antibodies can be screened for binding to NETs or theirability to disrupt NETs as described herein. These monoclonal antibodieswill usually bind with at least a KD of about 1 mM, more usually atleast about 300 μM, typically at least about 10 μM, more typically atleast about 30 Ian preferably at least about 10 μM, and more preferablyat least about 3 μM or better.

In some instances, it is desirable to prepare monoclonal antibodies fromvarious mammalian hosts, such as mice, rodents, primates, humans, etc.Description of techniques for preparing such monoclonal antibodies maybe found in, e.g., Stites, et al. (eds.) Basic and Clinical Immunology(4th ed.) Lange Medical Publications, Los Altos, Calif., and referencescited therein; Harlow and Lane (1988) Antibodies: A Laboratory ManualCSH Press; Goding (1986) Monoclonal Antibodies: Principles and Practice,(2d ed.) Academic Press, New York, N.Y.; and particularly in Kohler andMilstein (1975) Nature 256:495-497, which discusses one method ofgenerating monoclonal antibodies. Summarized briefly, this methodinvolves injecting an animal with an immunogen. The animal is thensacrificed any cells taken from its spleen, which are then fused withmyeloma cells. The result is a hybrid cell or “hybridoma” that iscapable of reproducing in vitro. The population of hybridomas is thenscreened to isolate individual clones, each of which secrete a singleantibody species to the immunogen. In this manner, the individualantibody species obtained are the products of immortalized and clonedsingle B cells from the immune animal generated in response to aspecific site recognized on the immunogenic substance.

Other suitable techniques involve selection of libraries of antibodiesin phage or similar vectors. See, e.g., Huse, et al. (1989) “Generationof a Large Combinatorial Library of the Immunoglobulin Repertoire inPhage Lambda,” Science 246:1275 1281; and Ward, et al. (1989) Nature341:544-546. The polypeptides and antibodies of the embodimentsdescribed herein may be used with or without modification, includingchimeric or humanized antibodies. Frequently, the polypeptides andantibodies will be labeled by joining, either covalently ornon-covalently, a substance which provides for a detectable signal. Awide variety of labels and conjugation techniques are known and arereported extensively in both the scientific and patent literature.Suitable labels include radionuclides, enzymes, substrates, cofactors,inhibitors, fluorescent moieties, chemiluminescent moieties, magneticparticles, and the like. Patents, teaching the use of such labelsinclude U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;4,277,437; 4,275,149; and 4,366,241. Also, recombinant immunoglobulinsmay be produced. See, Cabilly, U.S. Pat. No. 4,816,567; and Queen, etal. (1989) Proc. Nat'l Acad. Sci. USA 86:10029-10033.

Methods for developing small molecule, polymeric and genome basedlibraries are described, for example, in Ding, et al. J Am. Chem. Soc.124: 1594-1596 (2002) and Lynn, et al., J. Am. Chem. Soc. 123: 8155-8156(2001). Commercially available compound libraries can be obtained from,e.g., ArQule, Pharmacopia, graffinity, Panvera, Vitas-M Lab, BiomolInternational and Oxford. These libraries can be screened for ability todisrupt NETs using e.g. methods described herein.

In some embodiments, the anti-NET agent is a PAD4 inhibitor. As usedherein, “PAD4” refers to peptidylarginine deiminase 4, an enzyme thatconverts protein arginine residues to citrulline through a deiminationreaction (e.g. SEQ ID NO: 01 (mRNA) and SEQ ID NO: 02 (protein)).

PAD4 is distinguished from other PAD family enzymes by having a nuclearlocalization signal and thus being able to enter the nucleus andcitrullinate histones. As described herein, a loss of PAD4 activityresults in decreased NET formation and decreased DVT in mice. A PAD4inhibitor can decrease the expression or activity of PAD4.

Inhibition of PAD4 can be monitored by measuring PAD4 activity. Anon-limiting example of an assay of PAD4 activity is as follows: acandidate inhibitor, in a reaction buffer comprising 100 mM HEPES (pH7.6), 50 mM NaCl, and 0.5 mM tris(2-carboxyethyl)phosphine (TCEP) can bepreincubated with PAD4 (0.2 μM) (in the presence or absence of 10 mMCaCl2) at 37° C. for 15 min prior to the addition of the substrate,N-α-benzoyl-L-arginine ethyl ester (BAEE) (10 mM final concentration)(and 10 mM CaCl2 if CaCl2 was absent in the pre-incubation) to initiatethe reaction. After 15 min the reactions can be quenched by flashfreezing in liquid nitrogen. For color development, 200 μL of freshlyprepared COLDER solution (2.25 M H₃PO₄, 4.5 M H₂SO₄, 1.5 mM NH₄Fe(SO₄),20 mM diacetyl monoxime, and 1.5 mM thiosemicarbazide) can be added toeach of the quenched reactions, vortexed to ensure complete mixing, andthen incubated at 95° C. for 30 minutes. The absorbance at 540 nm canthen measured and compared to a citrulline standard curve to determinethe concentration of citrulline produced during the course of thereactions (PAD4 deaminates the BAEE substrate). IC50 values can bedetermined by fitting the concentration-response data to Eq. (1)

Fractional activity of PAD4=1/(1+([candidate inhibitor]/IC50)) (Eq. 1)The concentration of an inhibitor that corresponds to the midpoint(fractional activity=0.5) can be referred to as the IC50. Kits formeasuring PAD4 activity are also commercially available, e.g. Cat No.7000560, Cayman Chemical; Ann Arbor, Mich.

Any inhibitors of PAD4 can be used in the methods described herein. Forexample, in some embodiments, a PAD4 inhibitor can be a small moleculeinhibitor. Small molecule inhibitors of PAD4 are known in the art (see,for example, Luo et al. Biochemistry 2006; U.S. Pat. No. 7,964,636; andU.S. Patent Publications 2007/0276040 and 2011/0142868; each of which isincorporated by reference herein in its entirety) and include, by way ofnon-limiting example, Cl-amidine and F-amidine. In some embodiments, thePAD4 inhibitor can be specific for PAD4. In some embodiments, the PAD4inhibitor can be a PAD family inhibitor. PAD4 inhibitors arecommercially available, e.g. Cl-amidine (Catalog number 10599, CAS913723-61-2, Cayman Chemical; Ann Arbor, Mich.) and F-amidine (Catalognumber 10610; Cayman Chemica; Ann Arbor, Mich.).

As used herein, “Cl-amidine” refers to a compound having the structureof formula

As used herein, “Fl-amidine” refers to a compound having the structureof formula II:

In some embodiments, the PAD4 inhibitor can be an antibody, apolypeptide comprising a fragment of an antibody, or a nucleic acid.Antibodies, and methods of making them are described above herein. PAD4inhibitors which comprise a nucleic acid can be RNAi agents and/or genesilencing agents. As used herein, “gene silencing” or “gene silenced” inreference to an activity of an RNAi molecule, for example a siRNA ormiRNA refers to a decrease in the mRNA level in a cell for a target geneby at least about 5%, about 10%, about 20%, about 30%, about 40%, about50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99% ormore of the mRNA level found in the cell without the presence of themiRNA or RNA interference molecule. In one preferred embodiment, themRNA levels are decreased by at least about 70%, about 80%, about 90%,about 95%, about 99% or more.

As used herein, the term “RNAi” refers to any type of interfering RNA,including but are not limited to, siRNAi, shRNAi, endogenous microRNAand artificial microRNA. For instance, it includes sequences previouslyidentified as siRNA, regardless of the mechanism of down-streamprocessing of the RNA (i.e. although siRNAs are believed to have aspecific method of in vivo processing resulting in the cleavage of mRNA,such sequences can be incorporated into the vectors in the context ofthe flanking sequences described herein). The term “RNAi” and “RNAinterfering” with respect to an agent of the invention, are usedinterchangeably herein.

As used herein an “siRNA” refers to a nucleic acid that forms a doublestranded RNA, which double stranded RNA has the ability to reduce orinhibit expression of a gene or target gene when the siRNA is present orexpressed in the same cell as the target gene, sEH. The double strandedRNA siRNA can be formed by the complementary strands. In one embodiment,a siRNA refers to a nucleic acid that can form a double stranded siRNA.The sequence of the siRNA can correspond to the full length target gene,or a subsequence thereof. Typically, the siRNA is at least about 15-50nucleotides in length (e.g., each complementary sequence of the doublestranded siRNA is about 15-50 nucleotides in length, and the doublestranded siRNA is about 15-50 base pairs in length, preferably about19-30 base nucleotides, preferably about 20-25 nucleotides in length,e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides inlength).

As used herein “shRNA” or “small hairpin RNA” (also called stem loop) isa type of siRNA. In one embodiment, these shRNAs are composed of ashort, e.g. about 19 to about 25 nucleotide, antisense strand, followedby a nucleotide loop of about 5 to about 9 nucleotides, and theanalogous sense strand. Alternatively, the sense strand can precede thenucleotide loop structure and the antisense strand can follow.

The terms “microRNA” or “miRNA” are used interchangeably herein areendogenous RNAs, some of which are known to regulate the expression ofprotein-coding genes at the posttranscriptional level. EndogenousmicroRNA are small RNAs naturally present in the genome which arecapable of modulating the productive utilization of mRNA. The termartificial microRNA includes any type of RNA sequence, other thanendogenous microRNA, which is capable of modulating the productiveutilization of mRNA. MicroRNA sequences have been described inpublications such as Lim, et al., Genes & Development, 17, p. 991-1008(2003), Lim et al Science 299, 1540 (2003), Lee and Ambros Science, 294,862 (2001), Lau et al., Science 294, 858-861 (2001), Lagos-Quintana etal, Current Biology, 12, 735-739 (2002), Lagos Quintana et al, Science294, 853-857 (2001), and Lagos-Quintana et al, RNA, 9, 175-179 (2003),which are incorporated by reference. Multiple microRNAs can also beincorporated into a precursor molecule. Furthermore, miRNA-likestem-loops can be expressed in cells as a vehicle to deliver artificialmiRNAs and short interfering RNAs (siRNAs) for the purpose of modulatingthe expression of endogenous genes through the miRNA and or RNAipathways.

As used herein, “double stranded RNA” or “dsRNA” refers to RNA moleculesthat are comprised of two strands. Double-stranded molecules includethose comprised of a single RNA molecule that doubles back on itself toform a two-stranded structure. For example, the stem loop structure ofthe progenitor molecules from which the single-stranded miRNA isderived, called the pre-miRNA (Bartel et al. 2004. Cell 116:281-297),comprises a dsRNA molecule.

As used herein, the term “complementary” or “complementary base pair”refers to A:T and G:C in DNA and A:U in RNA. Most DNA consists ofsequences of nucleotide only four nitrogenous bases: base or baseadenine (A), thymine (T), guanine (G), and cytosine (C). Together thesebases form the genetic alphabet, and long ordered sequences of themcontain, in coded form, much of the information present in genes. MostRNA also consists of sequences of only four bases. However, in RNA,thymine is replaced by uridine (U).

As used herein, the term “nucleic acid” or “nucleic acid sequence”refers to any molecule, preferably a polymeric molecule, incorporatingunits of ribonucleic acid, deoxyribonucleic acid or an analog thereof.The nucleic acid can be either single-stranded or double-stranded. Asingle-stranded nucleic acid can be one strand nucleic acid of adenatured double-stranded DNA. Alternatively, it can be asingle-stranded nucleic acid not derived from any double-stranded DNA.In one aspect, the template nucleic acid is DNA. In another aspect, thetemplate is RNA. Suitable nucleic acid molecules are DNA, includinggenomic DNA, ribosomal DNA and cDNA. Other suitable nucleic acidmolecules are RNA, including mRNA, rRNA and tRNA. The nucleic acidmolecule can be naturally occurring, as in genomic DNA, or it may besynthetic, i.e., prepared based up human action, or may be a combinationof the two. The nucleic acid molecule can also have certain modificationsuch as 2′-deoxy, 2′-deoxy-2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl(2′-O-MOE), 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl(2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP),2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido(2′-O-NMA), cholesterol addition, and phosphorothioate backbone asdescribed in US Patent Application 20070213292; and certainribonucleoside that are is linked between the 2′-oxygen and the4′-carbon atoms with a methylene unit as described in U.S. Pat. No.6,268,490, wherein both patent and patent application are incorporatedhereby reference in their entirety.

In some embodiments, a nucleic acid which is or which encodes a PAD4inhibitor further comprises a vector. The term “vector”, as used herein,refers to a nucleic acid construct designed for delivery to a host cellor for transfer between different host cells. As used herein, a vectorcan be viral or non-viral. The term “vector” encompasses any geneticelement that is capable of replication when associated with the propercontrol elements and that can transfer gene sequences to cells. A vectorcan include, but is not limited to, a cloning vector, an expressionvector, a plasmid, phage, transposon, cosmid, chromosome, virus, virion,etc.

As used herein, the term “expression vector” refers to a vector thatdirects expression of an RNA or polypeptide from sequences linked totranscriptional regulatory sequences on the vector. The sequencesexpressed will often, but not necessarily, be heterologous to the cell.An expression vector may comprise additional elements, for example, theexpression vector may have two replication systems, thus allowing it tobe maintained in two organisms, for example in human cells forexpression and in a prokaryotic host for cloning and amplification. Asused herein, the term “viral vector” refers to a nucleic acid vectorconstruct that includes at least one element of viral origin and has thecapacity to be packaged into a viral vector particle. The viral vectorcan contain the PAD4 inhibitor in place of non-essential viral genes.The vector and/or particle may be utilized for the purpose oftransferring any nucleic acids into cells either in vitro or in vivo.Numerous forms of viral vectors are known in the art. Vectors useful inthe methods described herein can include, but are not limited to,plasmids, retroviral vectors, adenoviral vectors, adeno-associated viralvectors, and pox virus vectors.

The term “replication incompetent” when used in reference to a viralvector means the viral vector cannot further replicate and package itsgenomes. For example, when the cells of a subject are infected withreplication incompetent recombinant adeno-associated virus (rAAV)virions, the heterologous (also known as transgene) gene is expressed inthe patient's cells, but, the rAAV is replication defective (e.g., lacksaccessory genes that encode essential proteins for packaging the virus)and viral particles cannot be formed in the patient's cells. The term“transduction” as used herein refers to the use of viral particles orviruses to introduce exogenous nucleic acids into a cell. The term“transfection” as used herein in reference to methods, such as chemicalmethods, to introduce exogenous nucleic acids, such as the nucleic acidsequences encoding an agent which decreases the activity and/or level ofPAD4 as described herein, into a cell. As used herein, the termtransfection does not encompass viral-based methods of introducingexogenous nucleic acids into a cell. Methods of transfection includephysical treatments (electroporation, nanoparticles, magnetofection),and chemical-based transfection methods. Chemical-based transfectionmethods include, but are not limited to those that use cyclodextrin,polymers, liposomes, nanoparticles, cationic lipids or mixtures thereof(e.g., DOPA, Lipofectamine and UptiFectin), and cationic polymers, suchas DEAE-dextran or polyethylenimine.

Methods of making RNAi agents which inhibit the expression and/oractivity of PAD4 are well known in the art. Sequences complementary tothe mRNA encoding PAD4 (i.e. SEQ ID NO: 01) can be used to design RNAiagents as described above herein.

The disruption of NETS can be monitored in vivo or in vitro. In oneembodiment, the disruption of NETS is monitored by assessing the levelof NET release in stored blood in the presence and absence of a testcompound, e.g. by ELISA and/or determination of DNA concentration asdescribed herein. In one embodiment, the ability of a test compound todisrupt NETS is monitored in vivo, e.g. by determining the ability toprevent platelet adhesion and aggregation as described herein and/ordetermining the ability to prevent thrombosis or protect against strokeas described herein.

Treatment of Stored Blood Products

Certain embodiments are based on the further discovery by the inventorsthat NETs are present in stored blood products and that treatment ofstored blood products with DNase reduces the accumulation of NETs in theblood product. Accordingly, in certain embodiments, provided herein aremethods and devices for treating accumulations of NETs in stored bloodproducts in order to avoid NET-induced cytotoxicity and thrombosis. Incertain embodiments, provided herein are methods of preventing therelease and/or accumulation of NETs and/or degrading NETs in storedblood products with anti-NET compounds.

Increased storage time has been implicated in increased incidents ofmortality, pneumonia, post-injury multiple organ failure,hemorrheological disorders, serious infections, TRALI, and adversemicrocirculatory hemodynamics. Numerous studies have found thatleukodepletion of RBC components reduces the incidence of at leastnon-hemolytic transfusion reactions, graft-versus-host disease andposttransfusion purpura (Pruss et al., Transfusion and Apheresis Science2004 30:41-6; Williamson et al., Transfusion 2007 47:1455-67) andimproves outcomes in infants with Bordetella pertussis infections whoreceive transfusions (Rowlands et al., Pediatrics 2010 126:816-27).While leukodepletion is currently recognized as a best practice forblood transfusions, the costs involved are considerable and haveprevented this safety measure from being universally implemented (vanAken et al. Ned Tijdschr Geneeskd 2000 144:1033-6). Some embodimentsprovide a cost-effective means for decreasing the risk-benefit ratio ofusing stored blood products for transfusion by treatment of stored bloodproducts with at least one anti-NET compound.

In certain embodiments, the method provided herein is directed to thetreatment of stored blood products and comprises contacting the bloodproducts with an effective amount of at least one anti-NET compound. Theblood product to be contacted with an anti-NET compound can be wholeblood or a fraction of whole blood, e.g. plasma (platelet-rich orplatelet-poor plasma), platelets, or red blood cells.

The phrase “stored blood product,” as used herein, refers to a bloodproduct from a donor. In certain embodiments, the blood product will betransfused into a recipient. The blood product can be stored in a bloodcollection device or blood storage device for any given period of time,e.g. minutes, hours, days, weeks, up to months, prior to use and/ortransfusion. In one embodiment, the stored blood product is frozen. Inone embodiment, the stored blood product is not frozen.

In certain embodiments, the method provided herein is directed to thetreatment of stored blood products at the time the blood is collected.The stored blood product can be contacted with an anti-NET compoundduring manual or “on-line” collection of the blood product from a donor.During “on-line” collection (i.e. plateletpheresis, plasmapheresis, orleukapheresis), one blood component is removed and the remainingcomponents are returned to the donor during the course of the donationprocedure. The stored blood product can be contacted with an anti-NETcompound before, during, or after the separation process of “on-line”collection.

Ideally, all blood cell preparations should be from freshly drawn bloodand then immediately transfused to the recipient. Thus, in oneembodiment, the blood product is treated with an effective amount of atleast one anti-NET compound at the time of collecting the blood productfrom the donor and is shortly thereafter transfused to the recipient.

In one embodiment, the blood product is treated with an effective amountof at least one anti-NET compound at the time of collecting the bloodproduct (e.g. whole blood, plasma, platelets, RBCs) from the donor andis subsequently stored for later use.

In alternative embodiments, the stored blood product can be contactedwith an anti-NET compound at any point during storage of the bloodproduct. Whole blood is often separated into its components (e.g. redblood cells (RBCs), platelets, and plasma) for storage.

The stored blood products can be contacted with an anti-NET compoundbefore separation into components. In certain embodiments the storedblood products can be contacted with an effective amount of at least oneanti-NET compound during separation into components. Alternatively, thestored blood products can be contacted with an effective amount of atleast one anti-NET compound after separation into components. In certainembodiments, the stored blood products can be treated with an effectiveamount of at least one anti-NET compound prior to transfusion after theblood product has been stored for a period of time. In certainembodiments, an effective amount of at least one anti-NET compound canbe added to the blood product through the storage period, e.g. addeddaily or weekly while the blood product is being stored. In certainembodiments, an effective amount of at least one anti-NET compound isadded to RBC blood products which are stored at 1-6° C.

In certain embodiments, a composition containing at least one anti-NETcompound is used to reduce the prevalence of NETs in stored bloodproducts by at least 10%, least 20%, at least 40%, at least 50%, atleast 75%, at least 90%, or more. For example, a composition containingan anti-NET compound is used to reduce the prevalence of NETs in storedblood products, thereby reducing the occurrence, severity, duration, ornumber of symptoms associated with one of the following, which areoffered by way of example only; mortality, pneumonia, post-injurymultiple organ failure, hemorrheological disorders, infections, TRALI,and adverse microcirculatory hemodynamics. By “reduce” in this contextis meant a statistically significant decrease in such level. Thedecrease can be, for example, at least 10%, least 20%, at least 40%, atleast 50%, at least 75%, at least 90%, or more.

Any anti-NET compound can be used for treatment of stored bloodproducts. In some embodiments, the anti-NET compound is DNase. In someembodiments, the anti-NET compound is an inhibitor of PAD4.

An effective dose can also be determined by measuring a reduction inmortality, pneumonia, post-injury multiple organ failure,hemorrheological disorders, serious infections, TRALI, and adversemicrocirculatory hemodynamics in a population of patients receivingblood products treated with at least one anti-NET compound as comparedto patients receiving blood products not treated with an anti-NETcompound. The incidence or severity of any of these conditions can beused to determine the therapeutically effective dose. It is well withinthe ability of one skilled in the art to monitor efficacy of treatmentor prevention by measuring any one of such parameters, or anycombination of parameters.

Blood products can be treated with any effective amount of an anti-NETcompound. In one embodiment, blood products can be treated with about0.1 U/mL to about 5000 U/mL, i.e., about 0.1 U/mL, about 0.5 U/mL, about1 U/mL, about 5 U/mL, about 10 U/mL, about 20 U/mL, about 50 U/mL, about100 U/mL, about 500 U/mL, about 1000 U/mL, or about 5000 U/mL of DNaseor another anti-NET compound which is an enzyme. In one embodiment,blood products are treated with about 0.5 mg/kg, about 1.0 mg/kg, about2.0 mg/kg, about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, about 100mg/kg, about 500 mg/kg or about 1000 mg/kg of an anti-NET compound. Thetreatment of the blood product can be repeated, for example, on aregular basis, such as weekly (i.e., every week) for two weeks, threeweek, four weeks, five weeks, or six weeks or until the blood product isused. Treatment of the blood product with the anti-NET compound canreduce levels of a NETs by at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80% or at least 90% or more.

Some embodiments further relate to the use of at least one anti-NETcompound or a pharmaceutical composition thereof, e.g., for treatingstored blood to reduce NETs present therein, in combination with otherpharmaceuticals and/or other therapeutic methods, e.g., with knownpharmaceutical agents and/or known therapeutic methods, such as, forexample, those which can be beneficial to enhance the safety, efficacyor shelf-life of stored blood products. The anti-NET compound and anadditional agent can be administered in the same composition or theadditional agent can be administered as part of a separate compositionor by another method described herein.

Such agents can include anticoagulants or preservatives, for example,CPD, CP2D (Citrate Phosphate Double Dextrose), CPDA-1, CDP/ADSOL®,CDP/Optisol®, AS-3 (Additive Solution 3, Haemonetics Corp BraintreeMass.) and SAG-M. Pathogen inactivation technologies (PITs) can also beadded to blood products and include S-59, methylene blue, riboflavin,S-303, and PEN110 (Inactine®). In the case of multi-bag collection andstorage sets, additives can be contained in one or more of the bags ofthat set. In some embodiments, a composition for the treatment of storedblood products comprises an effective amount of at least one anti-NETcompound and an anticoagulant, preservative, or PIT.

In certain embodiments, the blood product can be contacted with ananti-NET compound, at the time of transfusing the stored blood productsinto a patient. In certain embodiments, at least one anti-NET compoundis administered to the transfusion patient separately from thetransfusion itself.

Some embodiments are directed to a device which contains an effectiveamount of at least one anti-NET compound. In certain embodiments thedevice is a blood collection device. In certain embodiments the deviceis a blood storage device. In certain embodiments the device is a blooddelivery device. Blood collection, storage, and delivery devices aretypically sterilized.

A “blood collection device” as used herein is any equipment used tocollect blood from a donor that will come into contact with the bloodproduct. In certain embodiments the blood collection device is a bloodcollection bag and/or one or more plastic satellite bags which areintegrally connected (e.g. a multiple blood bag system). In furtherembodiments the device can be, but is not limited to, needles,needle/catheter adapters, spike couplers, and tubes. Collection needlescan be ultra-thin walled or normal with a diameter ranging from 15 to 25gauge or similar. Blood collection tubes link the collection needle tothe blood bag(s) and connect to the bags via a jack, coupler or port inthe bag. Blood bags can be as described below. Filters can also beincorporated into blood collection devices in order to filter bloodduring the collection process, such as leukocyte filters for collectingleukocyte-reduced whole blood (Imuflex leukocyte reduction filter,Terumo Somerset, N.J.). In certain embodiments, the effective amount ofat least one anti-NET compound can be present within the filter, needle,bag, and/or blood collection tube.

A “blood storage device” as used herein is any equipment which willcontact a stored blood product during storage or manipulation of thestored blood product after collection and before transfusion. In certainembodiments, an effective amount of at least one anti-NET compound isprovided within a blood storage device. In certain embodiments, theblood storage device is a blood collection bag or bags. In certainembodiments, the blood storage device is a satellite bag. As describedherein, blood bags include, but are not limited to, primary or satelliteblood bags, typically having volumes of 200, 300, 450, 500 or 600 mL andtransfer blood bags typically having volumes of 75, 150, 300, 500, 600,800, 1000 or 2000 mL. In one embodiment, the blood storage device has avolume to accommodate at least 75 mL, e.g. at least 75 mL of bloodproduct, at least 100 mL of blood product, at least 200 mL of bloodproduct, at least 500 mL of blood product, at least 1000 mL of bloodproduct, at least 2000 mL of blood product or more. Bags can be providedas single, double, triple, quadruple, or quintuple bag sets which areconnected by sterile tubing. These bags can be equipped with ports,jacks, or couplers for connecting to other bags, tubing, or for removalof blood or blood products for sampling and testing. One or more of thesatellite bags can be a designated platelet storage bag. Multiple bagscan be linked via tubes and jacks, couplers and/or ports. Tubesassociated with blood collection or delivery bags can have a samplingpouch or diversion arm incorporated for the collection of small volumesof blood to be removed for testing. Such sampling pouches or diversionarms can be included such that the blood sample for testing is collectedbefore or after the primary volume of blood is collected. In certainembodiments, the effective amount of at least one anti-NET compound canbe present within the bag, bags, tubing, port, jack, coupler, plateletstorage bag, tubes, sampling pouches, and/or diversion arms.

A “blood delivery device” as used herein is any equipment used totransfer blood or a blood product from a storage device to the recipientthat will come into contact with the blood product. In certainembodiments, an effective amount of at least one anti-NET compound isprovided within a blood delivery device. Blood delivery devices caninclude, but are not limited to, tubes, needles, needle/catheteradapters, spike couplers, and filters commonly used for the delivery ofblood or blood products to a transfusion recipient. Included are 170,195, 200 micron or smaller mesh filters. In certain embodiments, theeffective amount of at least one anti-NET compound can be present withinthe tubes, needles, needle/catheter adapters, spike couplers, and/orfilters.

The anti-NET compound can be present in any of the devices comprisingblood collection devices, blood storage devices, or blood deliverydevices such that the blood product will be contacted with the anti-NETcompound during the normal procedures of collecting, storing, ortransfusing the blood product. Some embodiments are directed to a bloodbag comprising the bag and an anti-NET compound in the interior of thebag. Some embodiments are directed to a filter device comprising thefilter and at least one anti-NET compound. In certain embodiments, thefilter device can be integrated into a tube, port or jack of the bloodcollection, storage, and delivery device.

Methods for Treatment

Described herein are methods of treating conditions associated withNETs, the method comprising administering to a subject an effectiveamount of at least one anti-NET compound. As used herein, a condition“associated with NETs” can be a condition caused by NETs or aggravatedby NETs. Non-limiting examples of conditions associated with NETsinclude TRALI, acute lung injury, sickle cell disease, cancer, andcardiovascular conditions (e.g. _stroke; ischemic reperfusion;myocardial infarction; inflammation; thrombosis; and deep veinthrombosis).

In one embodiment, a method of preventing transfusion related acute lunginjury (TRALI) is provided. The method comprises contacting a storedblood product to be used for transfusion (i.e. a blood transfusionproduct) with an effective amount of at least one anti-NET compound. Insome embodiments, the methods described herein comprise providing aperson at risk for TRALI with an inhibitor of NETS, such as PAD4, priorto transfusion. As used herein, the term “preventing” as it relates toTRALI refers to amelioration of at least one measurable symptom ofTRALI. In one embodiment, the symptom is decreased by at least 10%, atleast 20%, at least 30%, at least 40%, or at least 50%.

In one embodiment, a method of treating TRALI is provided. The methodcomprises administering to a patient an effective amount of at least oneanti-NET compound. As used herein, “treating” as it relates to TRALIrefers to reducing at least one measurable symptom of TRALI. In oneembodiment, the method is for treating a patient with symptoms of TRALIfollowing a transfusion. In one embodiment, the symptom is decreased byat least 10%, at least 20%, at least 30%, at least 40%, or at least 50%.In one embodiment, the anti-NET compound is administered to thebloodstream. In one embodiment, the anti-NET compound is administeredintranasally.

TRALI is the leading cause of death among transfusion patients (Stubbsin Sadienberg et al. Transfusion Medicine Reviews 2010 24:305-324).Symptoms of TRALI include, but are not limited to, dyspnea, cyanosis,cough, fever, chills, bilateral pulmonary infiltration, pulmonary edemaand hypotension (Stubbs in Sadienberg et al. Transfusion MedicineReviews 2010 24:305-324). White blood cells have been associated withrisk for TRALI and leukodepletion is recommended as a means of reducingthe risk of TRALI. At least one study has correlated increased TRALIrisk with the age of the blood product (Benjamin in Sadienberg et al.Transfusion Medicine Reviews 2010 24:305-324). Studies in rats were ableto induce TRALI with plasma or lipid extracted from stored human RBCs orplatelets, but not from plasma or lipid from fresh human RBCs orplatelets (Looney in Sadienberg et al. Transfusion Medicine Reviews 201024:305-324). Some embodiments provide a means for reducing the incidenceand mortality of TRALI, i.e. treatment of stored blood products with atleast one anti-NET compound. Not wishing to be bound by theory,treatment of stored blood products with at least one anti-NET compoundcould circumvent the need to leukodeplete blood product for theprevention of TRALI or other transfusion-related complications such asshortness of breath, fever, etc.

In one embodiment, a method of treating acute lung injury is provided.The method comprises administering to a patient an effective amount ofat least one anti-NET compound. As used herein, the term “acute lunginjury” refers to a condition characterized by epithelial andendothelial cell perturbation and/or destruction, inflammatory cellinflux to the lung tissue, compromised gas exchange, surfactantdisruption, pulmonary edema, bilateral pulmonary infiltrates, normalcardiac filling pressures, atelectasis and potentially completerespiratory failure. Acute lung injury can be caused by a number offactors including, but not limited to, embolism, ischemia, hyperoxia,inflammation, sepsis, pancreatitis, oleic acid, acid aspiration, sepsis,oropharyngeal aspiration, lung infection, and/or exposure to ozone,polytetrafluoroethylene, nickel sulfate, and/or lipopolysaccharide.

As used herein, “treating” as it relates to acute lung injury refers toreducing at least one measurable symptom of acute lung injury. In oneembodiment, the symptom is decreased by at least 10%, at least 20%, atleast 30%, at least 40%, or at least 50%. In one embodiment, theanti-NET compound is administered to the bloodstream. In one embodiment,the anti-NET compound is administered intranasally.

Herein it is demonstrated that NETs represent a link betweeninflammation and thrombosis by providing a stimulus and scaffold forthrombus formation. In addition, markers of NETs were detected in athrombus and plasma of baboons subjected to deep vein thrombosis, anexample of inflammation-enhanced thrombosis. Furthermore, demonstratedherein is an increase in the level of circulating NETs duringcardiovascular stress, ischemia, and reperfusion. Identification of thelink of NETs to thrombosis provides NETs as a therapeutic target fortreating a variety of conditions involving thrombosis. As describedherein, we have determined that treatment of subjects with DNase oranti-histone antibodies reduces the level of NETs in the bloodstream andcan reduce the incidence and severity of stroke and deep veinthrombosis. Accordingly, provided herein are methods for treatingpatients to prevent the accumulation of harmful levels of NETs ordegrade existing levels of NETs, thus providing novel methods oftreating a number of cardiovascular conditions.

Certain embodiments provide a method for treating or preventing acardiovascular condition in a patient, i.e. administering atherapeutically effective dose of at least one anti-NET compound. Asused herein, the phrase “cardiovascular condition” is intended toinclude all disorders characterized by insufficient, undesired orabnormal cardiac function, e.g. ischemic heart disease, hypertensiveheart disease and pulmonary hypertensive heart disease, valvulardisease, congenital heart disease and any condition which leads tocongestive heart failure in a subject, particularly a human subject.Also included are any diseases and conditions of the blood vessels whichresult in insufficient, undesired, or abnormal cardiac function e.g.stroke, thrombosis, ischemia, ischemic reperfusion, vessel occlusion,inflammation etc. As used herein, “cardiovascular condition”, is notlimited to those conditions resulting from atherosclerosis. Insufficientor abnormal cardiac function can be the result of disease, injury and/oraging.

In certain embodiments, the method provided herein is directed to thetreatment or prevention of cardiovascular conditions caused orcontributed to, by NETs by administering to a patient an effective doseof an anti-NET compound. In a further embodiment the cardiovascularcondition is stroke, ischemic reperfusion, myocardial infarction,inflammation, and thrombosis. In some embodiments, the cardiovascularcondition can be induced by sickle cell disease.

In certain embodiments, the cardiovascular condition to be treated isthrombosis. Clinically, inflammation and infection are linked tothrombosis (Smeeth, L. et al. Lancet 2006 357:1075-9 and Wagner,Arterioscler Thromb Vasc Biol 2005 25:1321-4). Herein, evidence isprovided that NETs contribute to this link. Accordingly, someembodiments provide methods for treating or preventing thrombosis in apatient, e.g. methods for treating or preventing cardiovascularconditions complicated by thrombosis. Thrombosis is the occurrence of anclot in a blood vessel at a site of injury to the vessel or aninappropriate blood clot in a blood vessel and depends on the adhesion,activation, and aggregation of platelets. RBCs, whose function inthrombosis is not well defined, are especially abundant in venousthrombi. Final thrombus stability requires scaffolding provided by largepolymers, such as fibrin and von Willebrand factor (VWF). Deep veinthrombosis (DVT) is often linked to inflammation and infections. Acomplication of thrombosis is that the clot will detatch from the bloodvessel wall where it formed and lodge somewhere else in the circulatorysystem, blocking blood flow and causing an embolism.

In certain embodiments, the cardiovascular condition to be treated isischemia. In another embodiment, the cardiovascular condition to betreated is ischemic reperfusion. As used herein, the term “ischemia”refers to any localized tissue ischemia due to reduction of the inflowof blood. The flow of blood to a tissue can be reduced due to anabnormality in the blood vessels such as thrombosis, embolism, orvasoconstriction. The reduced flow of blood results in local anemia,reduced oxygen levels and eventually damage to the tissue. Ischemia canalso be caused by myocardial infarction, acute coronary syndrome,coronary artery bypass surgery, stroke, gastrointestinal ischemia,peripheral vascular disease, and surgical procedures. However, if bloodflow is restored (reperfusion) damage can still occur. For example,reperfused postischemic non-necrotic myocardium is poorly contractileand has reduced concentrations of high energy nucleotides, depressedsubcellular organelle function and membrane damage that resolves onlyslowly. A general concern with ischemic reperfusion is that when bloodflow returns, white blood cells will activate an inflammatory responseupon detecting the tissue damage caused by the ischemic conditions.Furthermore, the recruitment of leukocytes and/or platelets triggered bythe original tissue damage can restrict blood flow in smallercapillaries, resulting in a second wave of ischemia.

The term “myocardial ischemia” refers to a subset of ischemia thatencompasses circulatory disturbances caused by coronary atherosclerosisand/or inadequate oxygen supply to the myocardium. For example, an acutemyocardial infarction represents an irreversible ischemic insult tomyocardial tissue. This insult results in an occlusive (e.g., thromboticor embolic) event in the coronary circulation and produces anenvironment in which the myocardial metabolic demands exceed the supplyof oxygen to the myocardial tissue.

In certain embodiments, the cardiovascular condition to be treated ismyocardial infarction. A myocardial infarction (i.e. a heart attack) isthe death of heart muscle from the sudden blockage of a coronary arteryby a blood clot. Coronary arteries are blood vessels that supply theheart muscle with blood and oxygen. Blockage of a coronary arterydeprives the heart muscle of blood and oxygen, causing injury to theheart muscle. Injury to the heart muscle causes chest pain and pressure.Inflammation is known to contribute to development of a myocardialinfarction, particularly via formation of atherosclerotic plaques.Disruption of a plaque can cause thrombosis and lead to myocardialinfarction.

In certain embodiments, the cardiovascular condition to be treated isstroke. Eighty percent of strokes are ischemic, resulting from arterialocclusion of cerebral arteries whereas the remaining 20% are due tointracerebral hemorrhage. Thromboembolic occlusion of intracerebralarteries restricts downstream blood flow, promoting secondary thrombiformation within the cerebral microvasculature.

In certain embodiments, the cardiovascular condition to be treated isthrombosis and the patient has systemic lupus erythematosus (SLE). SLEis an autoimmune disease in which the immune system attacks thepatient's body, resulting in inflammation and tissue damage. DNA hasbeen detected on the cell surface of platelets from lupus erythematosuspatients (Frampton et al. Clin Exp Immunol 1986 63:621-8). Lupuspatients are also prone to develop venous thrombosis (Esmon, Blood Rev2009 23:225-9) and have a reduced ability to degrade NETs (Hakkim etal., PNAS 2010 107:9813-8). In one embodiment, the patient does not havelupus erythematosus.

In certain embodiments, the condition to be treated is sickle celldisease (SCD) is a condition in which RBCs are deformed and rigid. Thealtered RBCs are more likely to restrict blood flow at certain points inthe circulatory system, leading to a crisis. In SCD patients, a lethalcrisis is often precipitated by an infection. In light of the adhesionof RBCs to NETs, as described herein and in Fuchs et al. PNAS 2010107:15880-15885, anti-NET compounds are contemplated as a treatment forpreventing or treating painful occlusive ischemic events and/or apotentially lethal thromobitic event in SCD patients. In certainembodiments, administration of at least one anti-NET compound would beindicated by risk factors. Such risk factors can include, but are notlimited to, an infection or an inflammatory condition.

Some embodiments relate to the use of at least one anti-NET compound andcompositions containing at least one such anti-NET compound for thetreatment of a cardiovascular condition. The compositions describedherein can be administered to the patient before, during, or after theoccurrence of a cardiovascular event such as a stroke. For example, acomposition containing an anti-NET compound is used to reduce theseverity, duration, or number of symptoms associated with one of thefollowing, which are offered by way of example only; stoke, ischemicreperfusion, myocardial infarction, inflammation, lupus erythematosus,SCD and thrombosis. By “reduce” in this context is meant a statisticallysignificant decrease in such level. The decrease can be, for example, atleast 10%, at least 20%, at least 50%, at least 90% or more.

In some embodiments, the methods and compositions described hereinrelate to the treatment and/or prevention of deep vein thrombosis (DVT)in a subject, the method comprising administering to a subject aneffective dose of at least one anti-NET compound. In some embodiments,the formation of a deep vein thrombosis is prevented. In someembodiments, the progression of one or more signs or symptoms of DVT isprevented, e.g. a thrombus does not increase in size. In someembodiments, the severity of one or more signs or symptoms of DVT isdecreased, e.g. a thrombus decreases in size.

In some embodiments, the methods described herein relate to a method ofinhibiting the formation of NETs in a subject, the method comprisingadministering to a patient an effective dose of at least one anti-NETcompound. In some embodiments, inhibiting the formation of NETs cancomprise preventing the formation of a NET and/or reducing thelikelihood that a NET will form in a subject. In some embodiments,inhibiting the formation of NETs can comprise inhibiting the growth orprogression of pre-existing NETs and/or reducing the likelihood that apre-existing NET will grow or progress in a subject. In someembodiments, the method of inhibiting the formation of NETs can reducethe severity of symptoms associated with the development of NETs, e.g.thrombosis. In some embodiments, a subject receiving treatment toinhibit the formation of NETs can be a subject having or diagnosed ashaving a cardiovascular condition as described above herein. In someembodiments, a subject receiving treatment to inhibit the formation ofNETs can be a subject having or diagnosed as having a condition selectedfrom the group consisting of sickle cell disease, TRALI, and acute lunginjury. In some embodiments, a subject receiving treatment to inhibitthe formation of NETs can be a subject having or diagnosed as having acondition which makes the subject predisposed to thrombosis (i.e.prothrombotic). A condition which makes the subject prothrombotic can beany condition in which the subject is more likely to have or to form aNET as compared to a healthy subject. A non-limiting example of suchprothrombotic conditions includes cancer.

Some embodiments relate to the use of at least one anti-NET compound ora pharmaceutical composition thereof as a prophylactic for any of theconditions described herein. A patient exhibiting symptoms, markers, orindications of a condition described herein can be treated with at leastone anti-NET compound in order to prevent or reverse the progression ofthe condition or to lessen the severity of future symptoms, markers, orindicators of the condition.

In certain embodiments the methods provided herein involve the use of atleast one anti-NET compound. In further embodiments, the method providedherein involves the use of two or more anti-NET compounds.

In certain embodiments, the effective dose of at least one anti-NETcompound is administered to a patient repeatedly.

In certain embodiments, administering a single dose of an anti-NETcompound to a patient decreases the concentration of NETs in thebloodstream by at least 10%, e.g., by at least 20%, at least 30%, atleast 50%, at least 75%, at least 100%, at least 200% or more ascompared to the level of NETs prior to treatment with the anti-NETcompound.

In one embodiment, a single administration of an anti-NET compound to apatient decreases the level of an indicator, symptom, or marker of acondition described herein by at least 10%, e.g., by at least 20%, atleast 30%, at least 50%, at least 75%, at least 90% more as compared tothe level of the indicator, symptom, or maker of the condition prior totreatment with the anti-NET compound.

In one embodiment, a single administration of an anti-NET compound to agroup of patients decreases the incidence or severity of stroke,thrombosis, infarction, ischemia, lung injury or death by at least 10%,e.g., by at least 10%, by at least 20%, at least 30%, at least 50%, atleast 75%, at least 100%, at least 200% or more as compared to theincidence of sepsis, stroke, lung injury or death in a group of patientsnot administered the anti-NET compound.

In certain embodiments the composition comprising at least one anti-NETcompound further comprises a pharmaceutically acceptable carrier. Someembodiments, relate to the use of at least one anti-NET compound or apharmaceutical composition thereof, e.g., for treating a patient with acardiovascular condition, in combination with other pharmaceuticalsand/or other therapeutic methods, e.g., with known pharmaceutical agentsand/or known therapeutic methods, such as, for example, those which canbe beneficial to patients with a condition described herein.

In some embodiments, the further pharmaceutical agent can be ananti-thrombotic. Non-limiting examples of anti-thrombotics includeheparin, tPA (e.g. alteplase, reteplase, and tenecteplase),anistreplase, streptokinase, urokinase, coumadins (e.g. warfarin,acenocoumarol, phenprocoumonal, atromentin, and phenindione),idraparinux, fondaparinux, cyclooxygenase inhibitors (e.g. aspririn),adenosine diphosphate receptor inhibitors (e.g. clopidogrel, prasugrel,ticagrelor, and ticlopidine), phosphodiesterase inhibitors (e.g.cilostazol), glycoprotein IIB/IIA inhibitors (e.g. abciximab,eptifibatide, and tirofiban), adenosine reuptake inhibitors, (e.g.dipyridamole), and thromboxane receptor antagonists (e.g. terutroban).

Agents for treating a cardiovascular condition include, withoutlimitation, anti-inflammatory agents, anti-thrombotic agents and/orfibrinolytic agents, anti-platelet agents, lipid reducing agents, directthrombin inhibitors, glycoprotein IIb/IIIa receptor inhibitors andagents that bind to cellular adhesion molecules and inhibit the abilityof white blood cells to attach to such molecules (e.g. anti-cellularadhesion molecule antibodies), angiotensin converting enzyme inhibitor,angiotensin II receptor antagonists, calcium channel blockers,β-adrenergic receptor agonists, vasodilators, diuretics, α-adrenergicreceptor antagonists or an antioxidant. One agent which can be used toreduce the risk of a future myocardial disorder in an individual testingpositive for antibodies to a cardiac troponin is aspirin(acetylsalicylic acid). Another cardioprotective agent is PPADS(pyridoxal phosphate-6azophenyl-2′,4′-disulphonic acid).

Agents for treating lung injury include, without limitation, nitricoxide, anti-inflammatory agents, perfluorocarbon and replacementsurfactants. The anti-inflammatories used to treat acute lung injuryinclude, without limitation, ibuprofen, N-acetylcystein, procystein,ketaconazole, methylprednisone, pentoxifylline, and lysofylline.

A number of anti-inflammatory agents are known in the art, non-limitingexamples of which are Alclofenac; Alclometasone Dipropionate; AlgestoneAcetonide; Alpha Amylase; Amcinafal; Amcinafide; Amfenac Sodium;Amiprilose Hydrochloride; Anakinra; Anirolac; Anitrazafen; Apazone;Balsalazide Disodium; Bendazac; Benoxaprofen; Benzydamine Hydrochloride;Bromelains; Broperamole; Budesonide; Carprofen; Cicloprofen; Cintazone;Cliprofen; Clobetasol Propionate; Clobetasone Butyrate; Clopirac;Cloticasone Propionate; Cormethasone Acetate; Cortodoxone; Deflazacort;Desonide; Desoximetasone; Dexamethasone Dipropionate; DiclofenacPotassium; Diclofenac Sodium; Diflorasone Diacetate; Diflumidone Sodium;Diflunisal; Difluprednate; Diftalone, Dimethyl Sulfoxide; Drocinonide;Endrysone; Enlimomab; Enolicam Sodium; Epirizole; Etodolac; Etofenamate;Felbinac; Fenamole; Fenbufen; Fenclofenac; Fenclorac; Fendosal;Fenpipalone; Fentiazac; Flazalone; Fluazacort; Flufenamic Acid;Flumizole; Flunisolide Acetate; Flunixin; Flunixin Meglumine; FluocortinButyl; Fluorometholone Acetate; Fluquazone; Flurbiprofen; Fluretofen;Fluticasone Propionate; Furaprofen; Furobufen; Halcinonide; HalobetasolPropionate; Halopredone Acetate; Ibufenac; Ibuprofen; IbuprofenAluminum; Ibuprofen Piconol; Ilonidap; Indomethacin; IndomethacinSodium; Indoprofen; Indoxole; Intrazole; Isoflupredone Acetate;Isoxepac; Isoxicam; Ketoprofen; Lofemizole Hydrochloride; Lomoxicam;Loteprednol Etabonate; Meclofenamate Sodium; Meclofenamic Acid;Meclorisone Dibutyrate; Mefenamic Acid; Mesalamine; Meseclazone;Methylprednisolone Suleptanate; Morniflumate; Nabumetone; Naproxen;Naproxen Sodium; Naproxol; Nimazone; Olsalazine Sodium; Orgotein;Orpanoxin; Oxaprozin; Oxyphenbutazone; Paranyline Hydrochloride;Pentosan Polysulfate Sodium; Phenbutazone Sodium Glycerate; Pirfenidone;Piroxicam; Piroxicam Cinnamate; Piroxicam Olamine; Pirprofen;Prednazate; Prifelone; Prodolic Acid; Proquazone; Proxazole; ProxazoleCitrate; Rimexolone; Romazarit; Salcolex; Salnacedin; Salsalate;Salycilates; Sanguinarium Chloride; Seclazone; Sermetacin; Sudoxicam;Sulindac; Suprofen; Talmetacin; Talniflumate; Talosalate; Tebufelone;Tenidap; Tenidap Sodium; Tenoxicam; Tesicam; Tesimide; Tetrydamine;Tiopinac; Tixocortol Pivalate; Tolmetin; Tolmetin Sodium; Triclonide;Triflumidate; Zidometacin; Glucocorticoids; Zomepirac Sodium.

Anti-thrombotic and/or fibrinolytic agents include Plasminogen,prekallikrein, kininogens, Factors XII, XIIIa, plasminogen proactivator,tissue plasminogen activator[TPA], Streptokinase; Urokinase: AnisoylatedPlasminogen-Streptokinase Activator Complex; Pro-Urokinase; (Pro-UK);rTPA (alteplase or activase; r denotes recombinant), rPro-UK;Abbokinase; Eminase; Sreptase Anagrelide Hydrochloride; Bivalirudin;Dalteparin Sodium; Danaparoid Sodium; Dazoxiben Hydrochloride; EfegatranSulfate; Enoxaparin Sodium; Ifetroban; Ifetroban Sodium; TinzaparinSodium; tenecteplase, retaplase; Trifenagrel; Warfarin; Dextrans.

Anti-platelet agents include P2Y12 inhibitors, Clopridogrel;Sulfinpyrazone; Aspirin; Dipyridamole; Clofibrate; Pyridinol Carbamate;PGE; Glucagon; Antiserotonin drugs; Caffeine; Theophyllin Pentoxifyllin;Ticlopidine; Anagrelide. Lipid reducing agents include gemfibrozil,cholystyramine, colestipol, nicotinic acid, probucol lovastatin,fluvastatin, simvastatin, atorvastatin, pravastatin, cirivastatin.Direct thrombin inhibitors include hirudin, hirugen, hirulog, agatroban,PPACK, thrombin aptamers. Glycoprotein IIb/IIIa receptor inhibitors areboth antibodies and non-antibodies, and include but are not limited toReoPro (abciximab), lamifiban, eptifibatide and tirofiban.

Angiotensin converting enzyme inhibitors include captopril, enalapril,lisinopril, benazapril, fosinopril, quinapril, ramipril, spirapril,imidapril, and moexipril.

Angiotensin II receptor antagonists include both angiotensin I receptorsubtype antagonists and angiotensin II receptor subtype antagonists.Suitable antiotensin II receptor antagonists include losartan andvalsartan.

Calcium channel blockers include verapamil, diltiazem, nicardipine,nifedipine, amlodipine, felodipine, nimodipine, and bepridil.

Beta-adrenergic receptor antagonists include atenolol, propranolol,timolol, and metoprolol.

Vasodilators include hydralazine, nitroglycerin, and isosorbidedinitrate.

Diuretics include furosemide, diuril, amiloride, and hydrodiuril.

Alpha-adrenergic receptor antagonists include prazosin, doxazocin, andlabetalol.

Antioxidants include vitamin E, vitamin C, and isoflavones.

In certain embodiments for the treatment of stroke, tissue plasminogenactivator (tPA) or ADAMTS13, a molecule that cleaves von Willebrandfactor can be contemplated. In certain embodiments where a hemorrhageoccurs the agent can be an antihypertensive drug, e.g., a beta blockeror diuretic drug, a combination of a diuretic drug and apotassium-sparing diuretic drug, a combination of a beta blocker and adiuretic drug, a combination of an angiotensin-converting enzyme (ACE)inhibitor and a diuretic, an angiotensin-II antagonist and a diureticdrug, and/or a calcium channel blocker and an ACE inhibitor. In anothermore specific embodiment, the second therapeutic agent is a calciumchannel blocker, glutamate antagonist, gamma aminobutyric acid (GABA)agonist, an antioxidant or free radical scavenger.

Agents for treatment of lupus erythematosus include, but are not limitedto, nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids,antimalarials (i.e. chloroquinone), immunosuppressants (i.e.azathioprine or cyclophosphamide), heparin, aspirin, danazol(Danocrine), vincristine (Oncovin), warfarin, methylprednisolone pulsetherapy, dapsone, retinoids, thalidomide (Synovir); methylprednisolonesodium succinate (A-Methapred, Solu-Medrol), methotrexate (Rheumatrex),hydroxychloroquine (Plaquenil), or triamcinolone (Aristospan).

Agents for treatment of SCD include, but are not limited to,prophylactic antibiotics, acetaminophen, NSAIDs, hydroxyurea (Droxia,Hydrea), and agents which inhibit selectins or other adhesion molecules(e.g. TB-1269, OC-229, GMI-1070, and 3-(4-methoxybenzoyl)propionicacid).

The anti-NET compound and the pharmaceutically active agent can beadministrated to the subject in the same pharmaceutical composition orin different pharmaceutical compositions (at the same time or atdifferent times). When administrated at different times, an anti-NETcompound and the pharmaceutically active agent can be administeredwithin 5 minutes, 10 minutes, 20 minutes, 60 minutes, 2 hours, 3 hours,4, hours, 8 hours, 12 hours, 24 hours of administration of the other.When the anti-NET compound, and the pharmaceutically active agent areadministered in different pharmaceutical compositions, routes ofadministration can be different. For example, the anti-NET compound isadministered by any appropriate route known in the art including, butnot limited to oral or parenteral routes, including intravenous,intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary,nasal, rectal, and topical (including buccal and sublingual)administration, and pharmaceutically active agent is administration by adifferent route, e.g. a route commonly used in the art foradministration of said pharmaceutically active agent. In a non-limitingexample, an anti-NET compound can be administered orally, while apharmaceutically active agent can be administrated subcutaneously.

Efficacy of treatment or prevention of disease can be assessed, forexample by measuring a marker, indicator, or symptom of cardiovascularand/or pulmonary health or any other measurable parameter appropriate.It is well within the ability of one skilled in the art to monitorefficacy of treatment or prevention by measuring any one of suchparameters, or any combination of parameters.

A treatment is evident when there is a statistically significantimprovement in one or more parameters of cardiovascular and/or pulmonaryhealth, or by a failure to worsen or to develop symptoms where theywould otherwise be anticipated. As an example, a favorable change of atleast 10% in a measurable parameter of cardiovascular and/or pulmonaryhealth, and preferably at least 20%, 30%, 40%, 50% or more can beindicative of effective treatment. Efficacy for a given anti-NETcompound or formulation of that drug can also be judged using anexperimental animal model for a condition described herein as known inthe art. When using an experimental animal model, efficacy of treatmentis evidenced when a statistically significant increase in a marker isobserved.

The dosage ranges for the administration of an anti-NET compound dependupon the form of the compound, its potency, and the extent to whichsymptoms, markers, or indicators of a condition described herein aredesired to be reduced, for example the percentage reduction desired forapoptosis (necrosis), inflammation, or thrombus size. The dosage shouldnot be so large as to cause adverse side effects, such as hyperviscositysyndromes, pulmonary edema, congestive heart failure, and the like.Generally, the dosage will vary with the age, condition, and sex of thepatient and can be determined by one of skill in the art. The dosage canalso be adjusted by the individual physician in the event of anycomplication.

Patients can be administered a therapeutic amount of an anti-NETcompound, such as 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 5 mg/kg,10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg or 50 mg/kg.The anti-NET compound can be administered, for example, by intravenousinfusion over a period of time, such as over a 5 minute, 10 minute, 15minute, 20 minute, or 25 minute period. The administration is repeated,for example, on a regular basis, such as hourly for 3 hours, 6 hours, 12hours or longer or such as biweekly (i.e., every two weeks) for onemonth, two months, three months, four months or longer. After an initialtreatment regimen, the treatments can be administered on a less frequentbasis. For example, after administration biweekly for three months,administration can be repeated once per month, for six months or a yearor longer. Administration of the anti-NET compound can reduce levels ofa marker or symptom of a condition described herein, e.g., inflammationby at least 10%, at least 15%, at least 20%, at least 25%, at least 30%,at least 40%, at least 50%, at least 60%, at least 70%, at least 80% orat least 90% or more.

Before administration of a full dose of the anti-NET compound, patientscan be administered a smaller dose, such as a 5% infusion, and monitoredfor adverse effects, such as an allergic reaction.

Owing to the effects on cardiovascular and pulmonary health, acomposition according to the methods and compositions described hereinor a pharmaceutical composition prepared therefrom can enhance thequality of life.

Efficacy Measurement

The efficacy of a given treatment to reduce the negative effects of NETscan be determined by the skilled clinician. However, a treatment isconsidered “effective treatment,” as the term is used herein, if any oneor all of the signs or symptoms of a condition described herein arealtered in a beneficial manner, other clinically accepted symptoms areimproved, or even ameliorated, e.g., by at least 10% following treatmentwith a compound as described herein. Efficacy can also be measured by afailure of an individual to worsen as assessed by hospitalization, orneed for medical interventions (i.e., progression of the disease ishalted). Methods of measuring these indicators are known to those ofskill in the art and/or are described herein. Treatment includes anytreatment of a disease in an individual or an animal (some non-limitingexamples include a human, or a mammal) and includes: (1) inhibiting thedisease, e.g., preventing a stroke, myocardial infarction or crisisepisode; or (2) relieving the disease, e.g., causing regression ofsymptoms. An effective amount for the treatment of a disease means thatamount which, when administered to a mammal in need thereof, issufficient to result in effective treatment as that term is definedherein, for that disease. Efficacy of an agent can be determined byassessing physical indicators of, for example as described below.Efficacy can be assessed in animal models of a condition describedherein, for example treatment of a mouse following exposure to hypoxicconditions, and any treatment or administration of the compositions orformulations that leads to an decrease in the concentration of NETs inthe bloodstream or the decrease of at least one symptom, marker, orparameter of a cardiovascular condition.

Efficacy can be measured by a reduction in any of the symptoms of acondition described herein, for example, a reduction in inflammation(including swelling, redness, or heat) a reduction in ischemia, areduction in angina, a reduction in pain or a reduction in thrombusnumber and/or size. Efficacy can also be measured by a failure of anindividual to worsen as assessed by hospitalization or need for medicalinterventions (i.e., progression of the disease is halted or at leastslowed).

Another marker of the efficacy of treatment as described herein issurvival. Statistical survival rates for specific conditions describedherein are well established—when an individual or group of individualstreated according to the methods described herein survives beyond theexpected time or at a greater than expected rate, the treatment can beconsidered effective.

The efficacy of treatment according to the methods described herein canbe evaluated by following surrogate or indirect markers of cardiacand/or pulmonary health and function. For example, cardiac cell death,whether by necrosis or apoptosis, is generally accompanied by therelease of cardiac enzymes, including cardiac creatine kinase (CK).Assays for cardiac enzymes are routinely used in the diagnosis ofmyocardial infarction and can be used to monitor the efficacy of cardiacprotection according to the methods described herein. A decrease incardiac enzymes (e.g., a 10% or greater decrease), or a lower level thanis normally seen with an infarct of a given size, is indicative ofeffective treatment. Other markers include, for example, cardiacTroponin T (TnT), which is a marker of cardiac injury that is used as analternative for CK. In addition, one can perform “echocardiographicanalysis of ejection fraction” as a measure of cardiac injury,stabilization after injury or recovery after injury.

Other markers that can be determined include, one or more cardiactroponins, a natriuretic peptide or a natriuretic peptide-relatedmarker, an inflammation marker, D-dimer, cholesterol, homocysteine,adiponectin, sCD40L, myeloperoxidase, and ischemia modified albumin,markers of acute inflammation and so-called proximal inflammatorymarkers.

Acute inflammatory markers known to the person skilled in the artinclude C-reactive protein (CRP), fibrinogen, D-dimer, serum amyloid A(SAA), pregnancy-associated polypeptide A (PAPP-A), intercellularadhesion molecules (e.g. ICAM-1, VCAM-1), IL-1-beta, IL-6, IL-8, IL-17IL-18/IL-18b; TNF-alpha; myeloperoxidase (MPO); TF; monocytechemoattractant protein 1 (MCP-1); P-selectin; E-selectin; plateletactivating factor acetyl hydrolase (PAF-AH); von Willebrand Factor(vWF). Preferred markers of acute inflammation for use in a methoddescribed herein are CRP, fibrinogen, D-dimer and SAA, of which CRP andD-dimer are more preferably used. Proximal inflammatory markers aremacromolecules situated upstream, i.e. close to or at theethiopathogenetic origin of the disease event. In particular, they areproduced at the site of the coronary heart lesion, preferably at thesite of an arterial plaque. Proximal inflammatory markers are inparticular associated with the risk that plaques already present in anindividual will undergo inflammation, or growth, and with theprobability of plaque rupture and thrombus formation. Proximalinflammatory markers are known to the person skilled in the art, andnon-limiting examples include pregnancy-associated polypeptide A(PAPP-A), matrix metalloproteinases (MMPs, e.g. MMP-1, -2, -3, -4, -5,-6, -7, -9, -10, -11, -12) and lipoprotein-associated phospholipase A2(Lp-PLA2).

Other markers, such as anti-double stranded DNA antibodies for systemiclupus erythematosus, can also be used.

The skilled artisan will appreciate that there are many ways to use themeasurements of two or more markers in order to improve the diagnosticquestion under investigation. In a quite simple, but nonetheless ofteneffective approach, a positive result is assumed if a sample is positivefor at least one of the markers investigated. This can e.g. be the casewhen diagnosing an infectious disease, like AIDS, by either detecting anucleic acid or a polypeptide of the infectious agent or by detectingantibodies to the infectious agent. Frequently, however, the combinationof markers is mathematically/statistically evaluated. Preferably thevalues measured for markers of a marker panel, e.g. an antibody to acardiac troponin and the level of a cardiac troponin, are mathematicallycombined and the combined value is correlated to the underlyingdiagnostic question. Preferably the diagnostic question is the relativerisk of developing a cardiovascular condition in the future. Preferablythe relative risk is given in comparison to healthy controls. Preferablyhealthy controls are matched for age and other covariates.

Marker values can be combined by any appropriate state of the artmathematical method. Well-known mathematical methods for correlating amarker combination to a disease or to the risk of developing a diseaseemploy methods like, Discriminant analysis (DA) (i.e. linear-,quadratic-, regularized-DA), Kernel Methods (i.e. SVM), NonparametricMethods (i.e. k-Nearest-Neighbor Classifiers), PLS (Partial LeastSquares), Tree-Based Methods (i.e. Logic Regression, CART, Random ForestMethods, Boosting/Bagging Methods), Generalized Linear Models (i.e.Logistic Regression), Principal Components based Methods (i.e. SIMCA),Generalized Additive Models, Fuzzy Logic based Methods, Neural Networksand Genetic Algorithms based Methods. The skilled artisan will have noproblem in selecting an appropriate method to evaluate a markercombination as described herein. Preferably the method used incorrelating the marker combination described herein e.g. to the absenceor presence of myocardial disease is selected from DA (i.e. Linear-,Quadratic-, Regularized Discriminant Analysis), Kernel Methods (i.e.SVM), Nonparametric Methods (i.e. k-Nearest-Neighbor Classifiers), PLS(Partial Least Squares), Tree-Based Methods (i.e. Logic Regression,CART, Random Forest Methods, Boosting Methods), or Generalized LinearModels (i.e. Logistic Regression). Details relating to these statisticalmethods are found in the following references: Ruczinski, I., J. ofComputational and Graphical Statistics, 12 (2003) 475-511; Friedman, J.H., Regularized Discriminant Analysis, JASA 84 (1989) 165-175; Hastie,T., Tibshirani, R., Friedman, J., The Elements of Statistical Learning,Springer Series in Statistics, 2001; Breiman, L., Friedman, J. H.,Olshen, R. A., Stone, C. J., (1984) Classification and regression trees,California: Wadsworth; Breiman, L. Random Forests, Machine Learning, 45(2001) 5-32; Pepe, M. S., The Statistical Evaluation of Medical Testsfor Classification and Prediction, Oxford Statistical Science Series, 28(2003) and Duda, R. O., Hart, P. E., Stork, D. G., PatternClassification, Wiley Interscience, 2nd Edition (2001).

In some embodiments, an optimized multivariate cut-off is used for theunderlying combination of biological markers and to e. g. discriminatepatients with low, intermediate and high risk of developing acardiovascular condition. In this type of multivariate analysis themarkers are no longer independent but form a marker panel.

Accuracy of a diagnostic method is best described by itsreceiver-operating characteristics (ROC) (see especially Zweig, M. H.,and Campbell, G., Clin. Chem. 39 (1993) 561-577). The ROC graph is aplot of all of the sensitivity/specificity pairs resulting fromcontinuously varying the decision thresh-hold over the entire range ofdata observed.

The clinical performance of a laboratory test depends on its diagnosticaccuracy, or the ability to correctly classify subjects into clinicallyrelevant subgroups. Diagnostic accuracy measures the test's ability tocorrectly distinguish two different conditions of the subjectsinvestigated. Such conditions are for example health and disease orbenign versus malignant disease, respectively.

In each case, the ROC plot depicts the overlap between the twodistributions by plotting the sensitivity versus 1-specificity for thecomplete range of decision thresholds. On the y-axis is sensitivity, orthe true-positive fraction [defined as (number of true-positive testresults)/(number of true-positive+number of false-negative testresults)]. This has also been referred to as positivity in the presenceof a disease or condition. It is calculated solely from the affectedsubgroup. On the x-axis is the false-positive fraction, or 1-specificity[defined as (number of false-positive results)/(number oftrue-negative+number of false-positive results)]. It is an index ofspecificity and is calculated entirely from the unaffected subgroup.Because the true- and false-positive fractions are calculated entirelyseparately, by using the test results from two different subgroups, theROC plot is independent of the prevalence of disease in the sample. Eachpoint on the ROC plot represents a sensitivity/1-specificity paircorresponding to a particular decision threshold. A test with perfectdiscrimination (no overlap in the two distributions of results) has anROC plot that passes through the upper left comer, where thetrue-positive fraction is 1.0, or 100% (perfect sensitivity), and thefalse-positive fraction is 0 (perfect specificity). The theoretical plotfor a test with no discrimination (identical distributions of resultsfor the two groups) is a 45.degree. diagonal line from the lower leftcomer to the upper right comer. Most plots fall in between these twoextremes. (If the ROC plot falls completely below the 45.degree.diagonal, this is easily remedied by reversing the criterion for“positivity” from “greater than” to “less than” or vice versa.)Qualitatively, the closer the plot is to the upper left comer, thehigher the overall accuracy of the test.

One convenient goal to quantify the diagnostic accuracy of a laboratorytest is to express its performance by a single number. The most commonglobal measure is the area under the ROC plot. By convention, this areais always >0.5 (if it is not, one can reverse the decision rule to makeit so). Values range between 1.0 (perfect separation of the test valuesof the two groups) and 0.5 (no apparent distributional differencebetween the two groups of test values). The area does not depend only ona particular portion of the plot such as the point closest to thediagonal or the sensitivity at 90% specificity, but on the entire plot.This is a quantitative, descriptive expression of how close the ROC plotis to the perfect one (area=1.0).

Pharmaceutical Compositions

For administration to a subject or blood product, the compounds can beprovided in pharmaceutically acceptable compositions. Thesepharmaceutically acceptable compositions comprise atherapeutically-effective amount of at least one anti-NET compounddescribed above, formulated together with one or more pharmaceuticallyacceptable carriers (additives) and/or diluents. As described in detailbelow, the pharmaceutical compositions described herein can be speciallyformulated for administration in solid or liquid form, including thoseadapted for the following: (1) oral administration, for example,drenches (aqueous or non-aqueous solutions or suspensions), lozenges,dragees, capsules, pills, tablets (e.g., those targeted for buccal,sublingual, and systemic absorption), boluses, powders, granules, pastesfor application to the tongue; (2) parenteral administration, forexample, by subcutaneous, intramuscular, intravenous or epiduralinjection as, for example, a sterile solution or suspension, orsustained-release formulation; (3) topical application, for example, asa cream, lotion, gel, ointment, or a controlled-release patch or sprayapplied to the skin; (4) intravaginally or intrarectally, for example,as a pessary, cream, suppository or foam; (5) sublingually; (6)ocularly; (7) transdermally; (8) transmucosally; or (9) nasally.Additionally, compounds can be implanted into a patient or injectedusing a drug delivery system. Coated delivery devices can also beuseful. See, for example, Urquhart, et al., Ann. Rev. Pharmacol.Toxicol. 24: 199-236 (1984); Lewis, ed. “Controlled Release ofPesticides and Pharmaceuticals” (Plenum Press, New York, 1981); U.S.Pat. Nos. 3,773,919; 6,747,014; and U.S. Pat. No. 35 3,270,960.

As used here, the term “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

As used here, the term “pharmaceutically-acceptable carrier” means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, manufacturing aid (e.g.,lubricant, talc magnesium, calcium or zinc stearate, or steric acid), orsolvent encapsulating material, involved in carrying or transporting thesubject compound from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the patient. Some examples of materials which canserve as pharmaceutically-acceptable carriers include, but are notlimited to: (1) sugars, such as lactose, glucose and sucrose; (2)starches, such as corn starch and potato starch; (3) cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, methylcellulose,ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, suchas magnesium stearate, sodium lauryl sulfate and talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12)esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents,such as polypeptides and amino acids (23) serum component, such as serumalbumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23)other non-toxic compatible substances employed in pharmaceuticalformulations. Wetting agents, binding agents, fillers, lubricants,coloring agents, disintegrants, release agents, coating agents,sweetening agents, flavoring agents, perfuming agents, preservative,water, salt solutions, alcohols, antioxidants, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike can also be present in the formulation. The terms such as“excipient”, “carrier”, “pharmaceutically acceptable carrier” or thelike are used interchangeably herein.

Many organized surfactant structures have been studied and used for theformulation of drugs. These include monolayers, micelles, bilayers andvesicles. Vesicles, such as liposomes, have attracted great interestbecause of their specificity and the duration of action they offer fromthe standpoint of drug delivery. Liposomes are unilamellar ormultilamellar vesicles which have a membrane formed from a lipophilicmaterial and an aqueous interior. The aqueous portion contains thecomposition to be delivered. Liposomes can be cationic (Wang et al.,Biochem. Biophys. Res. Commun., 1987, 147, 980-985), anionic (Zhou etal., Journal of Controlled Release, 1992, 19, 269-274), or nonionic (Huet al. S.T.P.Pharma. Sci., 1994, 4, 6, 466). Liposomes can comprise anumber of different phospholipids, lipids, glycolipids, and/or polymerswhich can impart specific properties useful in certain applications andwhich have been described in the art (Allen et al., FEBS Letters, 1987,223, 42; Wu et al., Cancer Research, 1993, 53, 3765; Papahadjopoulos etal. Ann. N.Y. Acad. Sci., 1987, 507, 64; Gabizon et al. PNAS, 1988, 85,6949; Klibanov et al. FEBS Lett., 1990, 268, 235; Sunamoto et al. Bull.Chem. Soc. Jpn., 1980, 53, 2778; Illum et al. FEBS Lett., 1984, 167, 79;Blume et al. Biochimica et Biophysica Acta, 1990, 1029, 91; U.S. Pat.Nos. 4,837,028; 5,543,152; 4,426,330; 4,534,899; 5,013,556; 5,356,633;5,213,804; 5,225,212; 5,540,935; 5,556,948; 5,264,221; 5,665,710;European Patents EP 0 445 131 B1; EP 0 496 813 B1; and European PatentPublications WO 88/04924; WO 97/13499; WO 90/04384; WO 91/05545; WO94/20073; WO 96/10391; WO 96/40062; WO 97/0478).

The compositions described herein can be prepared and formulated asemulsions or microemulsions. Emulsions are typically heterogeneoussystems of one liquid dispersed in another in the form of dropletsusually exceeding 0.1 μm in diameter and have been described in the art.microemulsion can be defined as a system of water, oil and amphiphilewhich is a single optically isotropic and thermodynamically stableliquid solution and can comprise surfactants and cosurfactants. Both ofthese drug delivery means have been described in the art (see e.g.,Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins(8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 2, p. 199, 245, & 335; Higuchi et al., in Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301;Leung and Shah, in: Controlled Release of Drugs: Polymers and AggregateSystems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215;Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co.,Easton, Pa., 1985, p. 271; Constantinides et al., PharmaceuticalResearch, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin.Pharmacol., 1993, 13, 205; Ho et al., J. Pharm. Sci., 1996, 85, 138-143;Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991,p. 92; U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099).

In one embodiment, the liposome or emulsion formulation comprises asurfactant. Surfactants find wide application in formulations such asemulsions (including microemulsions) and liposomes. The nature of thehydrophilic group (also known as the “head”) provides the most usefulmeans for categorizing the different surfactants used in formulations(Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York,N.Y., 1988, p. 285). Suitable surfactants include fatty acids and/oresters or salts thereof, bile acids and/or salts thereof. In certainembodiments, the surfactant can be anionic, cationic, or nonionic. Theuse of surfactants in drug products, formulations and in emulsions hasbeen reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker,Inc., New York, N.Y., 1988, p. 285).

In some embodiments, various penetration enhancers can be employed toeffect the efficient delivery of anti-NET compounds across cellmembranes. Penetration enhancers can be classified as belonging to oneof five broad categories, i.e., surfactants, fatty acids, bile salts,chelating agents, and non-chelating non-surfactants all of which havebeen described elsewhere (see e.g., Malmsten, M. Surfactants andpolymers in drug delivery, Informa Health Care, New York, N.Y., 2002;Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991,p. 92; Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252; Touitou,E., et al Enhancement in Drug Delivery, CRC Press, Danvers, Mass., 2006;Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654;Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis ofTherapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996,pp. 934-935; Swinyard, Chapter 39 In: Remington's PharmaceuticalSciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa.,1990, pages 782-783; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263,25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583; Jarrett, J.Chromatogr., 1993, 618, 315-339; Katdare, A. et al., Excipientdevelopment for pharmaceutical, biotechnology, and drug delivery, CRCPress, Danvers, Mass., 2006; Buur et al., J. Control Rel., 1990, 14,43-51)

Oral formulations and their preparation are described in detail in U.S.Pat. No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No.6,747,014, each of which is incorporated herein by reference.Compositions and formulations for parenteral, intraparenchymal (into thebrain), intrathecal, intraventricular or intrahepatic administration caninclude sterile aqueous solutions which can also contain buffers,diluents and other suitable additives such as, but not limited to,penetration enhancers, carrier compounds and other pharmaceuticallyacceptable carriers or excipients. Aqueous suspensions can furthercontain substances which increase the viscosity of the suspensionincluding, for example, sodium carboxymethylcellulose, sorbitol and/ordextran. The suspension can also contain stabilizers.

A composition comprising at least one anti-NET compound can beadministered directly to the airways of a subject in the form of anaerosol or by nebulization. For use as aerosols, an anti-NET compound insolution or suspension may be packaged in a pressurized aerosolcontainer together with suitable propellants, for example, hydrocarbonpropellants like propane, butane, or isobutane with conventionaladjuvants. An anti-NET compound can also be administered in anon-pressurized form such as in a nebulizer or atomizer.

An anti-NET compound can also be administered directly to the airways inthe form of a dry powder. For use as a dry powder, an anti-NET compoundcan be administered by use of an inhaler. Exemplary inhalers includemetered dose inhalers and dry powdered inhalers.

Aerosols for the delivery to the respiratory tract are known in the art.See for example, Adjei, A. and Garren, J. Pharm. Res., 1: 565-569(1990); Zanen, P. and Lamm, J.-W. J. Int. J. Pharm., 114: 111-115(1995); Gonda, I. “Aerosols for delivery of therapeutic an diagnosticagents to the respiratory tract,” in Critical Reviews in TherapeuticDrug Carrier Systems, 6:273-313 (1990); Anderson et al., Am. Rev.Respir. Dis., 140: 1317-1324 (1989)) and have potential for the systemicdelivery of peptides and proteins as well (Patton and Platz, AdvancedDrug Delivery Reviews, 8:179-196 (1992)); Timsina et. al., Int. J.Pharm., 101: 1-13 (1995); and Tansey, I. P., Spray Technol. Market,4:26-29 (1994); French, D. L., Edwards, D. A. and Niven, R. W., AerosolSci., 27: 769-783 (1996); Visser, J., Powder Technology 58: 1-10(1989)); Rudt, S. and R. H. Muller, J. Controlled Release, 22: 263-272(1992); Tabata, Y, and Y. Ikada, Biomed. Mater. Res., 22: 837-858(1988); Wall, D. A., Drug Delivery, 2: 10 1-20 1995); Patton, J. andPlatz, R., Adv. Drug Del. Rev., 8: 179-196 (1992); Bryon, P., Adv. Drug.Del. Rev., 5: 107-132 (1990); Patton, J. S., et al., Controlled Release,28: 15 79-85 (1994); Damms, B. and Bains, W., Nature Biotechnology(1996); Niven, R. W., et al., Pharm. Res., 12(9); 1343-1349 (1995); andKobayashi, S., et al., Pharm. Res., 13(1): 80-83 (1996), contents of allof which are herein incorporated by reference in their entirety.

The compositions described herein can additionally contain other adjunctcomponents conventionally found in pharmaceutical compositions, at theirart-established usage levels. Thus, for example, the compositions cancontain additional, compatible, pharmaceutically-active materials suchas, for example, antipruritics, astringents, local anesthetics oranti-inflammatory agents, or can contain additional materials useful inphysically formulating various dosage forms of the compositionsdescribed herein, such as dyes, flavoring agents, preservatives,antioxidants, opacifiers, thickening agents and stabilizers. However,such materials, when added, should not unduly interfere with thebiological activities of the components of the compositions describedherein. The formulations can be sterilized and, if desired, mixed withauxiliary agents, e.g., lubricants, preservatives, stabilizers, wettingagents, emulsifiers, salts for influencing osmotic pressure, buffers,colorings, flavorings and/or aromatic substances and the like which donot deleteriously interact with the anti-NET compound(s) of theformulation.

As used herein, the phrase “subject in need of at least one anti-NETcompound” refers to a subject who is diagnosed with or identified assuffering from, having or at risk for developing a cardiovascularcondition or one or more complications related to a cardiovascularcondition.

A subject in need of at least one anti-NET compound can be identifiedusing any method used for diagnosis of, e.g. a cardiovascular condition.For example, Doppler ultrasonography can confirm a diagnosis of deepvein thrombosis. Parameters for diagnosis of cardiovascular conditionsare known in the art and available to skilled artisan without mucheffort.

In some embodiments, the methods described herein further compriseselecting a subject identified as being in need of an anti-NET compound.A subject in need of treatment with an anti-NET compound can be asubject having, diagnosed as having, or exhibiting the signs, symptoms,or markers of a condition associated with, caused by, or aggravated byNETs. Non-limiting examples of such conditions include, a cardiovascularcondition as described herein (e.g. stroke; ischemic reperfusion;myocardial infarction; inflammation; thrombosis; and deep veinthrombosis), sickle cell disease, TRALI, acute lung injury, and cancer.

A subject in need of an anti-NET compound can be selected based on thesymptoms presented, such as symptoms of stroke, thrombosis, or ischemia.Symptoms and markers of cardiovascular conditions are described herein.

Toxicity and therapeutic efficacy can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compositions that exhibit large therapeutic indices, are preferred.Murine genetics and surgical techniques have generated a number of mousemodels for the study of cardiovascular conditions or mice impaired inthe ability to limit the concentration of NETs. Such models can be usedfor in vivo testing of anti-NET compounds, as well as for determining atherapeutically effective dose. A suitable mouse model is, for example,the DNase^(−/−) mouse described herein or the mouse model of strokedescribed herein.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage can vary within this range depending upon the dosage formemployed and the route of administration utilized.

The therapeutically effective dose can be estimated initially from cellculture assays. A dose can be formulated in animal models to achieve acirculating plasma concentration range that includes the IC50 (i.e., theconcentration of the therapeutic which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Levels in plasmacan be measured, for example, by high performance liquid chromatography.The effects of any particular dosage can be monitored by a suitablebioassay.

The amount of an anti-NET compound which can be combined with a carriermaterial to produce a single dosage form will generally be that amountof the compound which produces a therapeutic effect. Generally out ofone hundred percent, this amount will range from about 0.1% to 99% ofcompound, preferably from about 5% to about 70%, most preferably from10% to about 30%.

The dosage can be determined by a physician and adjusted, as necessary,to suit observed effects of the treatment. Generally, the compositionsare administered so that the anti-NET compound is given at a dose from 1μg/kg to 150 mg/kg, 1 μg/kg to 100 mg/kg, 1 μg/kg to 50 mg/kg, 1 μg/kgto 20 mg/kg, 1 μg/kg to 10 mg/kg, 1 μg/kg to 1 mg/kg, 100 μg/kg to 100mg/kg, 100 μg/kg to 50 mg/kg, 100 μg/kg to 20 mg/kg, 100 μg/kg to 10mg/kg, 100 μg/kg to 1 mg/kg, 1 mg/kg to 100 mg/kg, 1 mg/kg to 50 mg/kg,1 mg/kg to 20 mg/kg, 1 mg/kg to 10 mg/kg, 10 mg/kg to 100 mg/kg, 10mg/kg to 50 mg/kg, or 10 mg/kg to 20 mg/kg. It is to be understood thatranges given here include all intermediate ranges, for example, therange 1 tmg/kg to 10 mg/kg includes 1 mg/kg to 2 mg/kg, 1 mg/kg to 3mg/kg, 1 mg/kg to 4 mg/kg, 1 mg/kg to 5 mg/kg, 1 mg/kg to 6 mg/kg, 1mg/kg to 7 mg/kg, 1 mg/kg to 8 mg/kg, 1 mg/kg to 9 mg/kg, 2 mg/kg to 10mg/kg, 3 mg/kg to 10 mg/kg, 4 mg/kg to 10 mg/kg, 5 mg/kg to 10 mg/kg, 6mg/kg to 10 mg/kg, 7 mg/kg to 10 mg/kg, 8 mg/kg to 10 mg/kg, 9 mg/kg to10 mg/kg etc. . . . It is to be further understood that the rangesintermediate to the given above are also within the scope of the methodsand compositions described herein, for example, in the range 1 mg/kg to10 mg/kg, dose ranges such as 2 mg/kg to 8 mg/kg, 3 mg/kg to 7 mg/kg, 4mg/kg to 6 mg/kg etc.

With respect to duration and frequency of treatment, it is typical forskilled clinicians to monitor subjects in order to determine when thetreatment is providing therapeutic benefit, and to determine whether toincrease or decrease dosage, increase or decrease administrationfrequency, discontinue treatment, resume treatment or make otheralteration to treatment regimen. The dosing schedule can vary from oncea week to daily depending on a number of clinical factors, such as thesubject's sensitivity to the anti-NET compound. The desired dose can beadministered at one time or divided into subdoses, e.g., 2-4 subdosesand administered over a period of time, e.g., at appropriate intervalsthrough the day or other appropriate schedule. Such sub-doses can beadministered as unit dosage forms. In some embodiments, administrationis chronic, e.g., one or more doses daily over a period of weeks ormonths. Examples of dosing schedules are administration daily, twicedaily, three times daily or four or more times daily over a period of 1week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months,5 months, or 6 months or more. The desired dose can be administeredusing continuous infusion or delivery through a controlled releaseformulation. In that case, the anti-NET compound contained in eachsub-dose must be correspondingly smaller in order to achieve the totaldaily dosage. The dosage unit can also be compounded for delivery overseveral days, e.g., using a conventional sustained release formulationwhich provides sustained release of the anti-NET compound over a severalday period. Sustained release formulations are well known in the art andare particularly useful for delivery of agents at a particular site,such as could be used with the agents described herein. In thisembodiment, the dosage unit contains a corresponding multiple of thedaily dose.

The skilled artisan will appreciate that certain factors can influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the anti-NET compounds described herein can bemade using conventional methodologies or on the basis of in vivo testingusing an appropriate animal model, as described elsewhere herein.

In some embodiments, pharmaceutical compositions described hereininclude (a) one or more anti-NET compounds and (b) one or morepharmaceutically effective compounds as described herein.

Assessing a Thrombotic Event

As described herein, the inventors have found that increased levels ofNETs are associated with and/or cause hypoxia, stroke, thrombosis,embolism, and lung injury. Accordingly, some embodiments are generallyrelated to assays and methods for assessing whether a patient hasexperienced or is likely to experience a thrombotic event. As usedherein, the term “thrombotic event” refers to a condition caused atleast in part, by blood clotting. Such conditions include but are notlimited to, stroke, embolism, ischemia, ischemic reperfusion, myocardialinfarction, thrombosis, TRALI or acute lung injury. In certainembodiments, the assays and methods are directed to determination of thelevel of NETs in a biological sample of a subject.

In certain embodiments, the subject may be exhibiting signs or symptomsof a thrombotic event. In certain embodiments, the subject may not beexhibiting signs or symptoms of a thrombotic event but be at risk ofdeveloping a thrombotic event due to transfusion, inflammation,infection or other risk factors described herein.

The methods and assays described herein include determining the level ofNETs in a sample obtained from a patient and comparing the amount of theNETs in the sample obtained from a patient to an amount of a reference,wherein if the amount of the NETs in the sample obtained from a patientis greater by a statistically significant amount from that of the amountof the reference, the subject has experienced a thrombotic event or islikely to experience a thrombotic event.

The sample obtained from a patient can include, but is not limited toblood or blood products. Blood products in the context of samplesobtained from a patient can include, but are not limited to, anycomponent of a patient's blood (e.g. plasma) and/or blood or a componentthereof that has been treated or processed (e.g. with an anti-coagulantor preservative).

In some embodiments, the reference can be the level of NETs in a normalhealthy subject with no symptoms or signs of a thrombotic event. Forexample, a normal healthy subject has normal cardiovascular andpulmonary lung function, has not received a transfusion, has no symptomsof inflammation, does not have an infection, and/or is not diagnosedwith one of the conditions described herein. The reference can also be acontrol sample, a pooled sample of control individuals or a numericvalue or range of values based on the same. In certain embodiments,wherein the progression of thrombotic event(s) or risk of thromboticevent(s) in a subject is to be monitored over time, the reference canalso be a sample taken from the subject at an earlier date. Thereference sample can be, but is not limited to blood or blood products.Blood products can include, but are not limited to, any component of asubject's blood (e.g. plasma) and/or blood or a component thereof thathas been treated or processed (e.g. with an anti-coagulant orpreservative).

In certain embodiments, the patient is determined to have experienced athrombotic event or be likely to experience a thrombotic event if thelevel of NETs in the sample obtained from the patient is greater by astatistically significant amount than the level of NETs in a reference.In certain embodiments, the patient is determined to have determined tohave experienced a thrombotic event or be likely to experience athrombotic event if the level of NETs in the sample obtained from thepatient is great by at least about 50%, at least about 75%, at leastabout 100%, at least about 200%, at least about 500%, at least about1000% or more.

Methods of determining the level of NETs in a sample are describedelsewhere herein. In certain embodiments, the level of NETs isdetermined using labeled DNA detection reagents (i.e. Hoechst 33258 orSytoxGreen), immunodetection of histones, detection of nucleosomesand/or components thereof (i.e. Cell death detection kit, Roche), orelectrophoresis of plasma DNA.

In certain embodiments, immunochemical techniques can be used.Immunochemistry is a family of techniques based on the use of anantibody, wherein the antibodies are used to specifically targetmolecules of interest (i.e. histones or double stranded-DNA). Theantibody typically contains a marker that will undergo a biochemicalreaction, and thereby experience a change color, upon encountering thetargeted molecules. In some instances, signal amplification can beintegrated into the particular protocol, wherein a secondary antibody,that includes the marker stain or marker signal, follows the applicationof a primary specific antibody.

Methods to measure the levels of target molecules detected byimmunochemical techniques are well known to a skilled artisan. Suchmethods to measure target molecule levels include ELISA (enzyme linkedimmunosorbent assay), western blot, immunoprecipitation,immunofluorescence using detection reagents such as an antibody orprotein binding agents. Alternatively, a peptide can be detected in asubject by introducing into a subject a labeled anti-peptide antibodyand other types of detection agent. For example, the antibody can belabeled with a radioactive marker whose presence and location in thesubject is detected by standard imaging techniques.

A Method of Assessing Efficacy of Anti-NET Treatments

As described herein, the inventors have found that increased levels ofNETs are associated with and/or cause hypoxia, stroke, thrombosis,embolism, and lung injury and have provided methods of treating orpreventing these disorders by administering one or more anti-NETcompounds. Accordingly, some embodiments are generally related to assaysand methods for assessing the efficacy of the administration of one ofmore anti-NET compounds. In certain embodiments, the assays and methodsare directed to determination of the level of NETs in a biologicalsample of a subject.

The methods and assays described herein include determining the level ofNETs in samples obtained from a patient before and after treatment withone or more anti-NET compounds, wherein a reduction in the level of NETsfollowing the treatment with the anti-NET compound is indicative ofefficacy.

The sample obtained from a patient can include, but is not limited to,blood or blood products. Blood products in the context of samplesobtained from a patient can include, but are not limited to, anycomponent of a patient's blood (e.g. plasma) and/or blood or a componentthereof that has been treated or processed (e.g. with an anti-coagulantor preservative).

In certain embodiments, the sample obtained from the patient prior totreatment with one or more anti-NET compounds can be obtained at anytime prior to administration of the anti-NET compound, for example,about 1 minute prior to treatment, about 10 minutes prior to treatment,about 1 hour prior to treatment, about 1 day prior to treatment, about 1week prior to treatment, about 2 weeks prior to treatment, about 1 monthprior to treatment, or earlier. In certain embodiments, the sampleobtained from the patient after treatment with one or more anti-NETcompounds can be obtained at any time after administration of theanti-NET compound, for example, about 10 minutes after treatment, about1 hour after treatment, about 1 day after treatment, about 1 week aftertreatment, about 2 weeks after treatment, or later.

In certain embodiments, the treatment is determined to have beenefficacious if the level of NETs after treatment is lower by astatistically significant amount than the level of NETs prior totreatment. In certain embodiments, the treatment is determined to havebeen efficacious if the level of NETs is reduced by at least about 10%,at least about 20%, at least about 30%, at least about 40%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90%, at least about 95%, at least about 98% or more.

Methods of determining the level of NETs in a sample are describedelsewhere herein.

Some embodiments of the technology described herein can be defined asany of the following numbered paragraphs:

-   1. A device which contains an effective amount of at least one    anti-NET compound wherein the device is selected from the group    consisting of: a blood collection device, a blood storage device,    and a blood delivery device.-   2. The device of paragraph 1, wherein the anti-NET compound is    DNase.-   3. The device of paragraph 1, wherein the anti-NET compound is a    PAD4 inhibitor.-   4. The device of paragraph 3, wherein the PAD4 inhibitor is selected    from the group consisting of:    -   Cl-amidine and F-amidine.-   5. The device of any of paragraphs 1-4 wherein the device is a blood    bag having an interior volume of at least 75 mL and not greater than    2000 mL.-   6. The device of any of paragraphs 1-5 wherein the device is a    filter contained in a tube which provides a means to move blood to    or from a blood bag.-   7. A method of treating stored blood products, comprising contacting    a blood product with an effective amount of at least one anti-NET    compound.-   8. The method of paragraph 7, wherein the anti-NET compound is    selected from the group consisting of:    -   DNase; RNAse; a histone-degrading enzyme; an inhibitor of        chromatin decondensation; an antibody against a component of a        NET; an elastase inhibitor; and a PAD4 inhibitor.-   9. The method of paragraph 8, wherein the PAD4 inhibitor is selected    from the group consisting of:    -   Cl-amidine and F-amidine.-   10. The method of any of paragraphs 7-9, wherein the blood product    is to be used for transfusion and is not frozen.-   11. The method of any of paragraphs 7-10, wherein the blood product    is contacted with an effective amount of an anti-NET compound at the    time of collecting said blood product from a donor.-   12. The method of any of paragraphs 7-11, wherein the effective    amount of an anti-NET compound is provided within a blood storage    device or blood collection device.-   13. The method of any of paragraphs 7-12, wherein the blood product    is contacted with an effective amount of an anti-NET compound while    the blood product is stored.-   14. The method of any of paragraphs 7-13, wherein the effective    amount of an anti-NET compound is provided within a blood storage    device.-   15. The method of any of paragraphs 7-14, wherein the blood product    is contacted with an effective amount of an anti-NET compound during    the process of transfusing said blood products into a patient.-   16. The method of any of paragraphs 7-15, wherein the effective    amount of an anti-NET compound is provided within a blood storage    device or a blood delivery device.-   17. The method of any of paragraphs 7-16, wherein the contacting    with an effective amount of an anti-NET compound occurs in vivo by    separate delivery of said blood product and said anti-NET compound    to the patient's bloodstream.-   18. The method of any of paragraphs 7-17, wherein the blood product    is selected from the group consisting of: whole blood, red blood    cells, blood plasma and platelets.-   19. A method of preventing transfusion-related acute lung injury    (TRALI), the method comprising contacting blood transfusion products    with an effective amount of an anti-NET compound.-   20. The method of paragraph 19, wherein the anti-NET compound is    selected from the group consisting of:    -   DNase; RNAse; a histone-degrading enzyme; an inhibitor of        chromatin decondensation; an antibody against a component of a        NET; an elastase inhibitor; and a PAD4 inhibitor.-   21. The method of paragraph 20, wherein the PAD4 inhibitor is    selected from the group consisting of:    -   Cl-amidine and F-amidine.-   22. A method of treating or preventing a condition associated with    NETs comprising administering to a patient an effective dose of at    least one anti-NET compound.-   23. The method of paragraph 22, wherein the anti-NET compound is    selected from the group consisting of:    -   DNase; RNAse; a histone-degrading enzyme; an inhibitor of        chromatin decondensation; an antibody against a component of a        NET; an elastase inhibitor; and a PAD4 inhibitor.-   24. The method of paragraph 23, wherein the PAD4 inhibitor is    selected from the group consisting of:    -   Cl-amidine and F-amidine.-   25. The method of any of paragraphs 22-24, wherein said condition is    a cardiovascular condition selected from the group consisting of:    -   stroke; ischemic reperfusion; myocardial infarction;        inflammation; thrombosis; and    -   deep vein thrombosis.-   26. The method of any of paragraphs 22-24, wherein said condition is    a condition selected from the group consisting of:    -   sickle cell disease, TRALI and acute lung injury.-   27. The method of any of paragraphs 22-26, wherein said effective    dose of anti-NET compound is administered prophylactically.-   28. The method of any of paragraphs 22-27, wherein said effective    dose of anti-NET compound is given repeatedly.-   29. The method of any of paragraphs 22-28, wherein the subject is    further administered an anti-thrombotic treatment.-   30. The method of paragraph 29, wherein the anti-thrombotic    treatment is selected from the group consisting of:    -   heparin; tPA; anistreplase; streptokinase; urokinase; a        coumadin; warfarin; idraparinux; fondaparinux; aspririn; a        adenosine diphosphate receptor inhibitor; a phosphodiesterase        inhibitor; a glycoprotein IIB/IIA inhibitor; a adenosine        reuptake inhibitor; and a thromboxane receptor antagonist.-   31. A method for treating or preventing deep vein thrombi in a    subject, the method comprising:    -   administering to a subject an effective dose of at least one        anti-NET compound.-   32. The method of paragraph 31, wherein the anti-NET compound is    selected from the group consisting of:    -   DNase; RNAse; a histone-degrading enzyme; an inhibitor of        chromatin decondensation; an antibody against a component of a        NET; an elastase inhibitor; and a PAD4 inhibitor.-   33. The method of paragraph 32, wherein the PAD4 inhibitor is    selected from the group consisting of:    -   Cl-amidine and F-amidine.-   34. The method of any of paragraphs 31-33, wherein said effective    dose of anti-NET compound is administered prophylactically.-   35. The method of any of paragraphs 31-34, wherein said effective    dose of anti-NET compound is given repeatedly.-   36. A method of any of paragraphs 31-35, wherein the subject is    further administered an anti-thrombotic treatment.-   37. The method of paragraph 36, wherein the anti-thrombotic    treatment is selected from the group consisting of:    -   heparin; tPA; anistreplase; streptokinase; urokinase; a        coumadin; warfarin; idraparinux; fondaparinux; aspririn; a        adenosine diphosphate receptor inhibitor; a phosphodiesterase        inhibitor; a glycoprotein IIB/IIA inhibitor; a adenosine        reuptake inhibitor; and a thromboxane receptor antagonist.-   38. A method of inhibiting the formation of NETs in a subject, the    method comprising administering to a patient an effective dose of at    least one anti-NET compound-   39. The method of paragraph 38, wherein the anti-NET compound is    selected from the group consisting of:    -   DNase; RNAse; a histone-degrading enzyme; an inhibitor of        chromatin decondensation; an antibody against a component of a        NET; an elastase inhibitor; and a PAD4 inhibitor.-   40. The method of paragraph 39, wherein the PAD4 inhibitor is    selected from the group consisting of:    -   Cl-amidine and F-amidine.-   41. The method of any of paragraphs 38-40, wherein the subject has a    cardiovascular condition selected from the group consisting of:    -   stroke; ischemic reperfusion; myocardial infarction;        inflammation; thrombosis; and    -   deep vein thrombosis.-   42. The method of any of paragraphs 38-41, wherein the subject has a    condition selected from the group consisting of:    -   sickle cell disease, TRALI and acute lung injury.-   43. The method of any of paragraphs 38-41, wherein the subject has    cancer.-   44. The method of any of paragraphs 38-43, wherein said effective    dose of anti-NET compound is administered prophylactically.-   45. The method of any of paragraphs 38-44, wherein said effective    dose of anti-NET compound is given repeatedly.-   46. The method of any of paragraphs 38-45, wherein the subject is    further administered an anti-thrombotic treatment.-   47. The method of paragraph 46, wherein the anti-thrombotic    treatment is selected from the group consisting of:    -   heparin; tPA; anistreplase; streptokinase; urokinase; a        coumadin; warfarin; idraparinux; fondaparinux; aspririn; a        adenosine diphosphate receptor inhibitor; a phosphodiesterase        inhibitor; a glycoprotein IIB/IIA inhibitor; a adenosine        reuptake inhibitor; and a thromboxane receptor antagonist.-   48. A method of assessing a thrombotic condition in a patient    comprising determining the level of NETs in a sample obtained from a    patient, wherein an increase in the level of NETs as compared to a    reference is indicative that a thrombotic event has occurred or is    likely to occur.-   49. A method of assessing the efficacy of the administration of an    effective dose of at least one anti-NET compound comprising    determining the level of NETs in a sample obtained from a patient    before and after treatment with the anti-NET compound, wherein a    reduction in the level of NETs following the treatment with the    anti-NET compound is indicative of efficacy.

EXAMPLES Example 1: NETs Promote Thrombosis

The interaction between NETs and blood was studied using NETs isolatedfrom human neutrophils. Blood samples were obtained from healthy donorswho had not taken any medication for at least 10 day. Platelets andneutrophils were prepared from ACD-blood (Brill A, et al. Cardiovasc Res2009 84(1):137-144) or EDTA-blood (Fuchs et al., J Cell Biol 2007176:231-241), respectively. Neutrophils were seeded into flow chambers(μ-Slide IV, IBIDI) at 0.5 to 1×10⁷ cells/mL. NET formation by themajority of cells was induced by phorbol 12-myristate 13-acetate (PMA,50 nM, 4 h; Sigma Aldrich) or glucose oxidase (GO, 1 U/mL, 4 h;Worthington Biochem), as previously described (Brill A, et al.Cardiovasc Res 2009 84(1):137-144). PMA-induced NETs were used forinitial observations. Other experiments were done with GO-induced NETs.NETs were washed and blocked with 1% BSA (Calbiochem). NET-DNA wasstained with 1 μg/mL Hoechst 33258 (Invitrogen) for 15 min at 37° C. orSytoxGreen (1 μM, Invitrogen). Washed platelets were loaded withfluorescent Calcein-AM (2.5 μg/mL, 10 min, 37° C.; Invitrogen) orplatelets in whole blood were labeled with Rhodamine 6G (5 μg/mL, 10min, 37° C.; Sigma). Fluorescent images were acquired by a ZeissAxiovert 200 inverted fluorescence microscope in conjunction with amonochrome camera (AxioCam MRm). Colors for fluorescence channels wereassigned using Axiovision software. Fluorescent areas in images werequantified using ImageJ software.

NETs were induced on glass coverslips and perfusion was performed usinga parallel-plate flow chamber system (Glycotech). NETs were perfusedwith platelets suspended in plasma at a shear rate of 200/s or 900/s and37° C. using a peristaltic pump and observed to be avidly adheringplatelets. NETs were washed and fixed with 2.5% glutaraldehyde andelectron microscopy was performed. Electron micrographs showed plateletaccumulation (labelled Pts in FIG. 1A) on a fibrous meshwork of NETs(FIG. 1A; scale bar=1 μm) and filopod formation indicated that platelets(labelled Pt in FIG. 1B) adherent on NETs were activated (FIG. 1B; scalebar=0.5 μm). NETs were also perfused with ACD-anticoagulated bloodsupplemented with the irreversible thrombin inhibitorPPACK-Dihydrochloride (100 μM; Calbiochem) and recalcified by additionof 2 mM CaCl₂. Perfusion at high shear rates (900/s) or low, typicallyvenous shear rates (200/s) resulted in time-dependent plateletaggregation. Cells firmly attached to collagen or NETs were lysed with100 μL of 0.5% Triton×100 in water. Hemoglobin content was measuredusing the method of Drabkin (Drabkin PNAS 1971 68:609-13). To quantifyplatelets, Rhodamine-6G fluorescence of the sample was analyzed using afluorometer.

Strings of NETs aligned in the direction of flow and, importantly, NETswere not a static surface but moved in three dimensions. Within 1 minfrom onset of perfusion, small platelet aggregates appeared on NETs.Platelet adhesion and aggregation on NETs increased over the next 9 min.DNase (100 U/mL; Worthington Biochem) simultaneously removed NETs andplatelets, indicating that platelets were indeed attached to NETs.Quantification showed that areas covered by NETs were constant (FIG. 1C;closed circles), whereas platelets adhered and aggregated in atime-dependent manner (FIG. 1D; closed circles). Both plateletaggregates and NETs were removed by DNase and 10 minutes after thebeginning of perfusion (FIGS. 1C and 1D). When blood was supplementedwith DNase at the beginning of the perfusion, NETs were degraded rapidly(FIG. 1C; open circles) and platelet aggregates did not form (FIG. 1D;open circles). Thus, NETs were the only prothrombotic substrate in theseexperiments. FIGS. 1C and 1D show data that are representative of atleast three independent experiments and are shown as mean±SEM, n=3. AUmeans arbitrary units.

Heparin (100 μg/mL; Sigma) almost completely dismantled NETs when theNETs were perfusion with heparinized blood (FIG. 2A). In addition,heparin (10 μg/mL, Sigma) removed platelet aggregates from NETs (FIG.2B) as efficiently as DNase (in FIGS. 2A-2B, data are presented asmean±SEM, n=3; Student's t test; *P<0.05; **P<0.01). The effect ofheparin was also observed in medium, indicating a direct interaction ofheparin with the NETs. Heparin has high affinity for histones (Pal etal., Thromb Res 1983 31:69-79) and releases histones from chromatin(Napirei et al. FEBS J 2009 276:1059-1073). Consequently, incubation ofNETs under static conditions with heparin or DNase alone releasedhistones to the culture supernatant (FIG. 2C; arrow indicates H2B;arrowhead may represent cross reactivity; DN=DNase). This resultindicates that heparin removes histones from the chromatin fibers thatbuilt the backbone of NETs and this leads to the destabilization ofNETs.

The possibility of a direct interaction between platelets and histoneswas examined. As shown in FIGS. 2D and 2E, histones were sufficient toinduce platelet aggregation. Incubation of platelets with histones H3 (5μg/mL; NEB; solid circles; FIG. 2E) and H4 stimulated aggregation,whereas histones H1, H2A, and H2B had no such effect (FIG. 2E). Thrombin(0.5 U/mL; open circles in FIG. 2D) and heparin (solid triangles in FIG.2D) served as a positive control in these experiments. Aggregation inresponse to histone H3 (FIG. 2D) and H4 (data not shown) was inhibitedby EDTA (5 mM; solid squares in FIG. 2D) which excluded plateletagglutination caused by the positive charge of histones. Heparincompletely abolished platelet response to these histones (FIG. 2E;ANOVA; ***P<0.001 compared with histone H1). Dissociating NETs andinhibiting histones could add to the antithrombotic effects of heparin.

When NETs were washed after perfusion with blood and observedmacroscopically a red thrombus was detectable (FIG. 3A). Shown in FIG.3A is a flow chamber coated with NETs after perfusion with blood. Lightmicroscopy of a red thrombus (arrow) anchored on two strings(arrowheads). FIG. 3A is a composite of multiple photographs of the flowchamber. (Scale bar, 500 μm). DNA staining revealed a scaffold of DNAand electron microscopy showed the presence of intact RBCs (FIG. 3B;scale bar, 5 μm). Quantification of RBC hemoglobin in flow chamberscoated with NETs or collagen and perfused with blood showed that RBCsbound to NETs but not collagen (FIG. 3C), although platelets bound toboth substrates (FIG. 3D). RBC adhesion to NETs was prevented when bloodwas supplemented with DNase, confirming that RBCs were attached to NETs.DNase had no effect on platelet adhesion to collagen-coated chambers,but prevented platelet adhesion to NETs (FIG. 3D). In summary, thesedata show that NETs provide a scaffold not only for platelets but alsofor RBC adhesion. Data presented are representative of at least threeindependent experiments and presented as mean±SEM, n=3; (ANOVA;**P<0.01); n.s.=not significant.

Whether NETs can concentrate plasma proteins that promote and stabilizethrombi (Frenette and Wagener NEJM 1996 335:43-5) was determined.Immunocytochemistry of NETs incubated with plasma showed that VWF andfibronectin, as well as fibrinogen, bound to NETs (FIGS. 19A-19C). Afterthe activation, NETs were incubated with 1% BSA for 1 h at 37° C. NETswere washed and incubated with 50% plasma in PBS for 30 min at 37° C.Next, NETs were washed and treated with DNaseI (100 U/mL, 10 min). Afteranother wash, cells were fixed with paraformaldehyde (2%, 1 h at 37° C.)and unspecific binding sites were blocked with BSA (3%, 1 h at 37° C.).Primary antibodies were used at 1 μg/mL in PBS supplemented with 1% BSAand 0.1% Triton×100 [mouse-antifibrinogen, rabbit-antifibronectin (bothSigma); rabbit-anti-VWF, (Chemicon)]. After incubation at 37° C. for 1h, samples were washed with PBS and fluorescently conjugated secondaryantibodies (Invitrogen) were applied at 10 μg/mL for 30 min at 37° C.These findings are corroborated by previous reports that VWF andfibrinogen interact with histones (Ward et al. Thromb Res 199786:469-77; Gonias et al., Thromb Res 1985 39:97-116) and thatfibronectin bears a DNA-binding domain (Pande et al., J Biol Chem 1985260:2301-6). The interaction of fibrinogen with NETs and its ability topromote fibrin deposition was determined. NETs were perfused withrecalcified blood supplemented with fluorescent fibrinogen (100 μg/mL;Invitrogen) and 20 mM CaCl₂ at the beginning of the perfusions.Fibrinogen was detected along NET-DNA strings and the depositiondrastically increased with perfusion time until the large fluorescentclot “embolized” together with the NETs. Thrombin was inhibited inparallel samples to prevent fibrinogen conversion to fibrin andpolymerization. Under these conditions, just traces of fibrinogen werefound on NETs and NETs remained stable during the entire perfusionperiod. Taken together, these experiments show that NETs supportplatelet-adhesion molecule deposition and thrombin-dependent fibrinformation.

The relative susceptibility of NETs and fibrin to serve as scaffolds forblood clots to thrombolysis was examined (FIG. 20). Neutrophils wereprestimulated with platelet-activating factor to release NETs withrecalcified blood under stirring conditions. Two-hundred-fiftymicroliters of 5×10⁶ neutrophils per milliliter in RPMI medium wereactivated by platelet activating factor (50 μM Calbiochem) at 37° C.under static conditions to induce NET formation. Control samples were250 μL medium alone, 250 μL of unstimulated neutrophils, or 250 μL of 50μM PAF in medium. After 10 min, 250 μL of recalcified (40 mM CaCl₂) ACDanticoagulated blood was added. The mixture was incubated under stirringconditions (1,000 rpm) at 37° C. using an Eppendorf Thermomixer.Indicated samples were supplemented with tPA (25 μg/mL, Baxter) or DNase1 (100 U/mL, Worthington Biochem). After 20 min, the blood was passedthrough a 100-μm cell strainer to isolate the clot. Images were acquiredand the clot was determined. Thereafter, 10-μm frozen sections of theclot were prepared and stained for fibrinogen(mouse-antihuman-fibrinogen, Sigma) and VWF (rabbit-antihuman-VWF,Chemicon). Isotype control antibodies were used to determine backgroundstaining and DNA was labeled fluorescently by Hoechst 33258(Invitrogen).

A single clot in which DNA intercalated with fibrin formed under theseconditions. Samples were treated with DNase to digest NETs or tissueplasminogen activator (tPA) for fibrin digestion. The tPA removed fibrinbut did not prevent clot formation. In tPA-resistant clots, RBCs andplatelets were held together by a DNA scaffold of NETs. Consequently,clot formation in the presence of activated neutrophils could beprevented only by simultaneous treatment with tPA and DNase. Thus, NETsmay provide a clot scaffold independent from fibrin.

Red thrombi, as well as leukocyte recruitment, are characteristics ofDVT (Esmon Blood Rev 2009 23:225-9). Thus, whether NETs are formed inexperimental DVT in baboons was determined. In brief, anesthetizedjuvenile male baboons underwent iliac vein thrombosis by temporaryballoon catheter occlusion (6 h). Six days postthrombosis, the animalwas humanely killed and both the thrombosed and nonthrombosed iliacveins were harvested. The iliac vein samples were then fixed andparaffin-embedded for immunohistochemical analysis.

Plasma was collected before and during DVT and analyzed for circulatingDNA (FIG. 4; BL=baseline), a marker of intravascular NET formation insepsis (Margraf et al., Shock 2008 30:352-8). Blood was drawn from theleft iliac vein using a 22-G vacutainer needle with a 4.5-mL sodiumcitrate vacutainer. After centrifugation at 3,350×g for 15 min at roomtemperature, aliquots were flash-frozen in liquid nitrogen and stored in−80° C. Time-points included: before (baseline), 6 h, 2 d, and 6 dpostthrombus induction. Plasma was diluted 10-fold in PBS and mixeddiluted plasma with an equal volume of 1 μM of the fluorescent DNA dyeSytoxGreen (Invitrogen) in PBS. Fluorescence was determined by afluorescence microplate reader (Fluoroskan, Thermo Scientific). Sampleswere normalized to the mean of values obtained from plasma collectedbefore induction of DVT (baseline).

Plasma DNA levels were low before and after the 6 h-DVT induction; thus,the surgical procedure did not increase this marker. Elevated plasma DNAlevels were detected 2 d after thrombus induction and remained increasedat 6 d postinduction (FIG. 4; bar represents the mean value of thegroups; Repeated measures ANOVA; **P<0.01 compared with BL). It isinteresting that the kinetics of the appearance of the fibrindegradation product D-dimer in plasma of baboons subjected to the samemodel is very similar (Meier et al., Thromb Haemost 2008 99:343-51).

Baboon DVT was also analyzed using a blood vessel staining kit(Millipore) with a different set of antibodies. The following primaryantibodies were used: mouse-antihistone H2A/H2B/DNA complex (42),rabbit-anti-VWF (Chemicon), and rabbit-antihistone H3 (Abeam). Primaryand isotype control antibodies were employed at 1 μg/mL; fluorescentlyconjugated secondary antibodies (Invitrogen) at 10 μg/mL DNA was labeledwith Hoechst 33342 (1 μg/mL, Invitrogen), or SytoxGreen (1 μM,Invitrogen).

DNA staining of the thrombosed iliac vein showed the circular vesselwall and within the lumen a dispersed punctuate staining, indicatingnuclei from leukocytes as well as a dense DNA core. This image comprisedtwo distinct DNA patterns: the dotted staining of nuclei and a diffusestaining of extracellular DNA, reminiscent of NETs. Positive stainingusing an antibody specific for DNA/histone complex showed that the DNAwas of nuclear rather than mitochondrial origin. Immunolocalization ofVWF revealed abundant VWF strings within the DNA core and in the areabetween the DNA core and the vessel wall. The DNA pattern oftenoverlapped with that of VWF. As a control, the right iliac vein from thesame baboon was analyzed. No indications of NETs were observed in thistissue. Areas within the thrombus lacking visible extracellular DNA wereabundant in histones, indicating the degradation of extracellular DNApresumably by nucleases in plasma. In summary, markers of NETs arepresent in plasma and within the thrombus of baboons subjected to DVT.

NETs are a unique link between inflammation and thrombosis which providea stimulus and scaffold for thrombus formation and markers of NETs areabundant in DVT. Inhibition of leukocyte infiltration in the baboonmodel of DVT produces unstable thrombi (Meier et al., Thromb Haemost2008 99:343-51). One way leukocytes can promote thrombus stability is byproducing NETs. The results described herein show that NETs colocalizewith fibrin in vitro.

Ischemia results in the production of IL-8 and reactive oxygen species.IL-8 is capable of inducing NETs (Brinkmann et al., Science 2004303:1532-5) and is considered a risk factor for venous thrombosis. Invitro stimulation of neutrophils with exogenous reactive oxygen speciesis sufficient to induce NETs (Fuchs et al., J Cell Biol 2007176:231-41). Mechanistically, NETs provide a scaffold for platelet andRBC adhesion and concentrate effector proteins involved in thrombosis.

DNA has been detected on the cell surface of platelets from patientswith systemic lupus erythematosus (Frampton et al., Clin Exp Immunol1986 63:621-8). Lupus patients are prone to develop venous thrombosis(Esmon Blood Rev 2009 23:225-9) and were recently described to haveimpaired NET degradation (Hakkim et al. PNAS 2010 107:9813-8). RBCadhesion to NETs could also play a role in sickle-cell disease, where alethal crisis is often precipitated by infection (Booth et al., Int JInfect Dis 2010 14:e2-e12).

Statistical analysis included mean±SEM, ANOVA, Student's t test, andrepeated measures ANOVA. Results were considered significant at P<0.05.

Example 2: NETs in Blood Storage Products

In order to determine if some of the toxicity associated withnon-leukocyte depleted blood products was due to the accumulate of NETsduring blood storage, the levels of NETs in blood bank samples weredetermined. Plasma DNA concentrations of two blood bags, one withleukocytes and one depleted of leukocytes (leuko-reduced) weredetermined. Blood was stored in the blood bank at Brigham and Women'sHospital & Dana Faber Cancer Institute, Boston for more than 42 days inblood transfusion bags. Plasma was separated from blood cells by twocentrifugations. Blood was centrifuged at 3000 g for 10 min. Theplatelet poor plasma was collected and spun again at 10000 g for 5 min.Aliquots of double-centrifuged plasma were stored at −80° C. Plasma wasthen diluted 1:10 in phosphate buffered saline (PBS). Fifty μl ofdiluted plasma was mixed with 50 μl of PBS containing 2 μM SytoxGreen(Invitrogen) to label DNA and fluorescence was recorded in a fluorometer(Fluoroskan, Thermo Fisher Scientific). Auto-fluorescence was consideredas background and determined in samples mixed with PBS withoutSytoxGreen. Plasma from non-leukoreduced blood had high levels of plasmaDNA (4255 ng/ml) compared to plasma from leuko-reduced blood (138ng/ml). These results indicated that leukocytes release DNA into plasmaduring blood storage.

During a second round of experimentation, the blood bank providedadditional blood bags. Quantification of plasma DNA showed that theaverage DNA levels in 10 non-leukoreduced blood (Non-R in FIGS. 5A-5B)were approximately 100-fold higher that in plasma of fresh blood takenfrom 10 healthy donors (control) (FIG. 5A). Low levels of DNA weredetected in plasma from 14 bags depleted of leukocytes (Leuko-R in FIG.5A). These experiments suggest that leukocytes commonly release highlevels of NETs during blood storage.

The release of NETs in stored blood was examined by detecting histones.An ELISA assay (Cell death detection kit; Roche) was used in which twoantibodies are employed; one which will recognize histones H2A, H2B, H3,and H4 (all of which are present in NETs) and a second antibody specificfor double-stranded DNA. One unit of nucleosomes was defined as theaverage amount of nucleosomes quantified in plasma from fresh bloodcontrols. Again, stored plasma from blood bags containing leukocytes(Non-R) showed higher levels of NETs that fresh blood (control) orstored leukocyte-depleted plasma (Leuko-R) (FIG. 5B). This confirmed theconclusion that leukocytes release NETs during storage of bloodproducts.

In order to rule out the possibility that apoptotic cells were releasingDNA, total plasma DNA from three samples of non-leukocyte depleted bloodwas subjected to gel electrophoresis. DNA was isolated from plasma usinga DNA isolation kit according to manufacturer's instructions (Omegabio-tek, Norcross, Ga.), subjected to 2% agarose gel electrophoresis inthe presence of ethidium bromide and visualized using a geldocumentation system (BioRad, Hercules, Calif.). Apoptotic DNA is highlydegraded, whereas the DNA associated with NETs is not degraded.Electrophoresis of the plasma DNA revealed undegraded, high-molecularweight DNA, confirming that earlier assays were detecting NETs and notapoptotic DNA.

In order to determine if an anti-NET compound could be used to treatstored blood products, the effects of DNaseII on NET release duringblood storage was tested. Blood collected from healthy donors was storedfor up to three days and mixed with Citrate-Phosphate-Dextrose (CPD) toprevent coagulation. Blood was stored at room temperature on a rotarymixer. During storage NET release into plasma was quantified by theELISA (FIG. 6). If blood was supplemented with 10 U/mL DNaseII(Worthington Biochem), no NETs were detected in plasma. These resultsindicated that NETs can be degraded by an anti-NET compound during bloodstorage. It is noteworthy that blood is stored in the absence ofdivalent cations and at acidic pH. Most nucleases require calcium andmagnesium ions to degrade DNA and would therefore not function in storedblood. DNaseII cleaves DNA without divalent cations and functionsoptimally at low pH.

In all cases statistical analysis included mean±SEM, analysis ofvariance (ANOVA). Results were considered significant at P<0.05.

Example 3: Anti-NET Compounds in the Treatment of Stroke

Data are expressed as mean plus or minus SEM. All statistical analysiswas performed using Prism 4 (version 4.0b, Graphpad Software, Inc., LaJolla, Calif.). Infarct volumes and neurologic scores were analyzedusing the unpaired 2-tailed Student t test. P values less than 0.05 wereconsidered statistically significant.

In order to determine if NETs were released during cardiovascularstress, the level of NETs was determined in plasma samples of wild-typeC57BL/6 mice kept under normoxic (room air) and hypoxic (hypoxia chamberwith 6% 02) conditions for 24 hours. Wild type C57Bl/6 mice werepurchased form Jackson Laboratory (Bar Harbor, Me.). All animals used inthis work were 8-10 weeks old males (except for hypoxia experiments inwhich females were used). Animals were provided with free access tostandard laboratory chow and water and were kept on a light/dark cycleof 12 h. Mice were exposed to normobaric hypoxia at 6% oxygen for 24hours or were housed at normal air room pressure. For hypoxia treatment,animals were placed in a controlled atmosphere animal chamber(A-15274-P, Biospherix, Lacona, N.Y.). Hypoxia was achieved bysubstituting nitrogen for oxygen using a Pro:ox model 110 compact oxygencontroller (Biospherix, Lacona, N.Y.). Hypoxic animals had significantlyincreased nucleosome levels that were approximately 5 times higher thannormoxic mice.

Next, the question of whether cerebral ischemia/reperfusion injuryeffects circulating levels of NETs was addressed. Wild-type C57BL/6 micewere subjected to 2 hours of transient middle cerebral artery occlusion(tMCAO), followed by 22 hours of reperfusion. Focal cerebral ischemiawas induced by 60 or 120 min tMCAO. Mice were anesthetized with 2%isoflurane/oxygen mixture. Following a midline skin incision in theneck, the proximal common carotid artery, and the external carotidartery were ligated, and a standardized silicon rubber-coated 6.0 nylonmonofilament (6021; Doccol Corp., Redlands, Calif.) was inserted andadvanced via the right internal carotid artery to occlude the origin ofthe right MCA. Operation time per animal did not exceed 15 minutes. Theintraluminal suture was left in situ during the complete occlusion time.Then animals were re-anesthetized, and the occluding monofilament waswithdrawn to allow reperfusion. In animals undergoing a sham treatment,the exact same procedure was followed except that the monofilament wasonly inserted three quarters of the normal distance that is necessary toocclude the MCA, after which it was immediately withdrawn. Some animalswere exclusively used for laser-Doppler flowmetry (Periflux 5000,Perimed, Kings Park, N.Y.) to monitor regional cerebral blood flow(rCBF) in the MCA territory.

Before the surgical procedure and after reperfusion, blood samples werecollected and plasma was prepared. Sham-operated animals underwent thesame surgery but without occlusion of the right MCA. Levels ofcirculating DNA and nucleosomes were determined in the plasma samples(FIGS. 7A-7D). To measure nucleosome and DNA levels in plasma, blood wascollected from the retro-orbital sinus using 0.5M EDTA as anticoagulant.Plasma was prepared by centrifuging anticoagulated whole blood for 5 minat 2300×g. Plasma supernatant was carefully removed and centrifugedagain for 5 min at 2300×g to remove any contamination with blood cells.The plasma was stored at −80° C. until analysis. Nucleosome levels weremeasured using the Cell Death Detection ELISA^(PLUS) (Roche,Indianapolis, Ind.). Plasma was diluted 1:10 (V:V) in phosphate bufferedsaline. Fifty μl of diluted plasma was mixed with 50 μl of PBScontaining SytoxGreen (final concentration 2 μM, Invitrogen) to labelDNA and fluorescence was recorded in a fluorometer (Fluoroskan, ThermoFisher Scientific). Auto-fluorescence was considered as background anddetermined in samples mixed with phosphate buffered saline withoutSytoxGreen. FIGS. 7A and 7B show the levels of extracellular nucleosomesin, respectively, sham and stroke-induced animals. FIGS. 7C and 7D showthe levels of extracellular DNA in, respectively, sham andstroke-induced animals. The sham procedure increased both nucleosome andDNA levels. This 3.07±0.79 and 1.32±0.16 fold over baseline levelsrespectively most likely reflects cell death caused by the invasivesurgery. However, in animals that experienced stroke, levels ofcirculating nucleosomes and DNA were significantly more increased to7.65±1.85 and 4.88±1.68 times baseline levels respectively (p<0.05).These results show that cerebral ischemia/reperfusion injury results inthe generation of NETs.

As shown herein, the ability of DNase-1 to cleave DNA/platelet stringslimits the extent of extracellular DNA trap-mediated platelet adhesionand aggregation. It was therefore hypothesized that this enzyme mayprevent excessive platelet adhesion and aggregation induced byextracellular chromatin generated by ischemic stroke. To test the roleof DNase-1 in stroke, DNase-1^(−/−) mice were used in the tMCAO model.DNase1⁻¹⁻ mice were generated in a mixed 129×C57BL/6 genetic backgroundand back-crossed into the pure C57BL/6 genetic background for tengenerations. The gene knockout was performed by classical geneticmethods including the physical mapping of the gene locus by restrictionsite analysis, identification of the gene locus at chromosome 16 byFISH-analysis and isolation of a HindIII genomic DNA-fragment from abacteriophage library. At the time when the DNase1 knockout wasgenerated and published no other gene was described to be located in thevicinity of the DNase1 gene locus. However, due to the sequencing of themurine genome it became clear recently that the deletion of the DNase1gene affects another gene located partly in the 3′-untranslated (3′-UTR)and -flanking region (3′-FR) of the DNase1 gene. The original geneknockout scheme was amended by the location of exons 13 to 18 of theTrap1/Hsp75 (TNF type I receptor-associated protein 1/heat shock protein75) gene, whose reading-frame is located on the opposite DNA-strand thanthat of the DNase1 gene. Due to the deletion of a 2886 Bp longEcoRI/SmaI genomic DNA-fragment of the DNase1 gene locus parts of thecoding region and the complete 3′-UTR of exon 18 of the Trap 1 gene weredeleted in addition.

The cerebral vasculature of WT and DNase-1^(−/−) mice showed no majoranatomic differences that could influence stroke outcome (FIG. 8A, 8B).For assessment of the cerebral vasculature, animals were deeplyanesthetized with isoflurane and transcardially perfused with phosphatebuffered saline, followed by 5 ml black ink. Brains were carefullyremoved, fixed in 4% PFA and the Circle of Willis and major arterieswere examined under a dissecting microscope. The development of theposterior communicating arteries (PComAs) was examined and scored asdescribed (Murakami et al. Stroke 1997 28: 1797-1804). The circle ofWillis and the distribution of the MCA trunk and branch appeared to beanatomically identical between the genotypes. In addition, the scoreassessing formation of the posterior communicating arteries of bothhemispheres, which can influence susceptibility to tMCAO, did not differsignificantly in WT and DNase-1^(−/−) mice (1.80±0.37 versus 1.60±0.24respectively, p>0.05, FIG. 8A). No differences were found in theregional cerebral blood flow (rCBF) in the right MCA territory (FIG.8B). WT and DNase-1^(−/−) mice showed a comparable decrease in rCBFduring tMCAO (7.71±3.74% of baseline versus 10.27±0.65% of baselinerespectively, p>0.05) and a comparable degree of reperfusion (76.82±6.51of baseline versus 74.04±7.69% of baseline respectively, p>0.05). Nodifferences in cell blood counts could be detected between the twogenotypes (FIG. 8C).

Interestingly, DNase-1^(−/−) mice were more prone to ischemic stroke(FIGS. 9A-9B). Wild type (WT) and DM/se-1^(−/−) mice were subjected to 1h of tMCAO and 23 h of reperfusion after which mice and brains wereanalyzed. Mice were killed 24 hours after initiation of tMCAO. Brainswere quickly removed and cut into 2-mm-thick coronal sections using amouse brain slice matrix. The slices were stained with 2%2,3,5-triphenyl-tetrazolium chloride (TTC; Sigma-Aldrich, St. Louis,Mo.) in PBS to visualize the infarctions. Sections were photographedwith a digital Nikon D70 camera and infarct areas (white) were measuredblindly using Image J software (National Institutes of Health). Braininfarct volumes were measured by planimetry (FIG. 9A). Compared to thewild-type controls, DNase-1^(−/−) mice developed larger braininfarctions (84.10±7.24 mm³ versus 58.50±4.04 mm³, p<0.05, FIG. 9A).

This difference in infarct size was functionally relevant as both theBederson score and the grip test (FIG. 9B) score were significantlyworse in DNase-1^(−/−) mice (*P<0.05). Neurological function wasassessed, blinded for the mouse genotype 24 h, after initiation oftMCAO, using the modified Bederson score (Bederson et al., Stroke 198617:472-6). This test determines global neurological function accordingto the following scoring system: 0, no deficit; 1, forelimb flexion; 2,decreased resistance to lateral push; 3, unidirectional circling; 4,longitudinal spinning; 5, no movement. The grip test was performed asdescribed (Moran et al., PNAS 1995 92:5341-5). A mouse was placed on awooden bar (3 mm in diameter, 40 cm long) that is attached to twovertical supports 40 cm above a flat surface. When placing the mouse ata point midway between the supports, the experiment was rated accordingto the following system: 0, falls off; 1, hangs onto bar by twoforepaws; 2, as for 1, but attempts to climb onto bar; 3, hangs onto barby two forepaws plus one or both hindpaws; 4, hangs onto bar by all fourpaws plus tail wrapped around bar; 5, escape (where mouse was able towork its way to one of the supports). To assess behavior in the cornertest (Zhang et al., J Neurosci Methods 2002 117:207-214), a mouse wasplaced on a flat surface between two vertical boards that are arrangedin an angle of 30° with a small opening along the joint between the twoboards to encourage entry into the corner. The mouse was placed betweenthe two angled boards facing the corner and half way to the corner. Whenentering deep into the corner, the mouse rears forward and upward, thenturns back to face the open end. The non-ischemic mouse turns eitherleft or right, but the ischemic mouse preferentially turns toward thenon-impaired, ipsilateral (right) side. The turns in one versus theother direction were recorded from ten trials for each test. Turningmovements that were not part of a rearing movement were not scored. Micethat were not able to walk (Bederson score 4 or 5) were excluded. Thesedata indicate that DNase-1 can have a protective effect in ischemicstroke. Since DNase-1 deficiency is accompanied in this strain by areduced expression of TRAP-1, it was decided to corroborate the resultsby infusion of recombinant human DNase-1 (rhDNase-1) in wild-type mice.

To further investigate the possible protective role of DNase-1 inischemic stroke, WT animals were treated with recombinant human DNase-1(rhDNase-1)(Dornase alpha, Pulmozyme®, Genentech Inc. San Francisco,Calif.) during a 1 h tMCAO and 23 h of reperfusion. Fifteen minutesbefore surgery, 50 μg of rhDNase-1 was given intra-peritoneally and thiswas repeated 12 h later. In addition, five minutes before reperfusion,10 μg of rhDNase-1 was given via retro-orbital intravenous injection.Infarct size and volume was determined as described herein. Compared tovehicle-treated animals, mice receiving rhDNase-1 developedapproximately 40% smaller infarcts (81.45±9.08 mm³ versus 50.67±8.74mm³, p<0.05, FIG. 10A.). Treatment of mice with rhDNase-1 also led todramatic improvement in functional outcome as shown by tests assessingneurologic and motoric behavior. Compared to vehicle-treated mice, theBederson test score, the grip test score and the corner test result wereall significantly better in rhDNase-1 treated mice (p<0.05, FIG. 10B).

Nucleosomes, key constituents of NETs, are basically segments of DNAwrapped around a histone protein core. Extracellular histones have beenshown to be cytotoxic toward endothelium, to be able to induceintravascular thrombosis and even to cause death when administered insufficient amount to mice. To assess whether extracellular histones arealso of pathological significance in the progression of ischemic stroke,histone-neutralizing antibody (BWA3, 10 mg/kg) was infused 5 min beforereperfusion (FIGS. 12A-B). Antibodies against histone H4 were isolatedfrom cell culture supernatants of hybridoma clone BWA3 by affinitychromatography on protein G columns. Purified antibodies were dialyzedagainst saline and were characterized by testing for binding to purifiedhistones H1, H2A, H2B, H3, H4 and a mixture of all histones in an ELISAset-up. BWA3 antibodies bound to H2A and H4 and consequently also to thehistone mix. IgG1 did not bind to histones (FIG. 11). Administration ofthe histone-neutralizing antibody resulted in a protective effect: whencompared to animals treated with either vehicle or IgG1 isotype controlantibody, infarct volumes of BWA3 treated mice were significantlysmaller (46.00±5.82 mm³ versus 68.15±6.58 mm³ (vehicle, p<0.05) or63.43±4.29 mm³ (isotype control, p<0.05. FIG. 12A). Treatment withhistone-neutralizing antibody (BWA3) consisted of a single intravenousretro-orbital bolus injection of antibody (BWA3 or IgG1 isotype controlantibody) at a concentration of 10 mg/kg five minutes beforereperfusion. Neurologic/motoric outcome as measured by the Bederson andgrip test score (FIG. 12B) was better in BWA3-treated mice, reachingstatistical significance for the grip test score (compared tovehicle-treated animals). The protective effect of thishistone-neutralizing antibody indicates that histones (H4 and H2B) are amajor mediator of ischemic stroke in mice.

The data shown herein indicate that NETs are increased after ischemicstroke. DNase-1 appears to have a protective role in ischemic strokesince the absence of DNase-1 aggravates stroke outcome whereas infusionof rDNase-1 improves stroke outcome. Moreover, neutralization ofhistones using an anti-histone antibody also had a protective effect inthis stroke model. Taken together, the data suggest an important role ofNETs in the development of ischemic stroke.

Experimental ischemic stroke results in a significant increase of bothDNA and nucleosome levels in the circulation. Interestingly,significantly elevated concentrations of DNA and nucleosomes have alsobeen found in stroke patients (Geiger et al., Cerebrovascular Diseases2006 21:32-7; Lam et al., Resuscitation 2006 68:71-8; Rainer et al.,Clin Chem 2003 49:562-9; Tsai et al., Clinica chimica acta 2001412:476-9). In these patients, DNA/nucleosome levels correlated stronglywith stroke severity and were associated with morbidity, mortality anddegree of disability. In one study, comparison with other biomarkerssuch as S100 protein, neuron-specific enolase, C-reactive protein andleukocytes, nucleosomes on day 3 after stroke were the only independentprognostic biomarker for recovery after one year (Geiger et al.,Cerebrovascular Diseases 2006 21:32-7). Liberation of chromatin and itsdegradation products from damaged cells seems a plausible mechanism forincreased DNA/nucleosome levels during and after stroke. However, withincreased circulating DNA/nucleosome levels as a non-specific cell deathindicator and with ischemic cell damage being considered to be a dynamicprocess with considerable inter-individual variation, the strikingassociation between circulating DNA/nucleosome levels and strokeseverity has always been surprising. Inflammatory responses, inparticular the recruitment of neutrophils, can lead to significantchromatin release by forming NETs. Indeed, while stroke causesneutrophilia, and an increase in the number of circulating monocytes(Wang et al., J Neuroimmunol 2007 184:53-68) a recent study shows thatthe level of plasma DNA in patients with acute ischemic strokecorrelates positively with white blood cell count (Tsai et al., Clinicachimica acta 2001 412:476-9). In addition, myeloperoxidase levels, amarker for neutrophil activation, were found to be increased afterstroke (Barone et al., J Neurosci Res 1991 29:336-345; Barone et al.,Molecular and chemical neuropathology 1995 24:13-30; Cojocaru et al.,Romanian Journal of Internal Medicine 2010 48:101-4). It is wellestablished that inflammatory responses and recruitment of neutrophilsplay an important role in the pathophysiology of ischemic stroke.Neutrophil depletion, inhibition of neutrophil adhesion and inhibitionof neutrophil function are all strategies that have been shown to reduceinfarct volume and improve stroke outcome. The detrimental effect ofneutrophils has classically been attributed to the no-reflow phenomenon(obstruction of the microcirculation) and release of toxic agents suchas oxygen free radicals and proteolytic enzymes. However, as shownherein, NETs released by neutrophils also promote thrombosis and bindred blood cells. These NETs, formed by a special cell-death program thatinvolves internal membrane dissolution, chromatin decondensation andcytolysis (Fuchs et al., Journal of Cell Biology 2007 176:231-241), areable to adhere, activate and aggregate platelets as shown herein.Interestingly, besides neutrophils, also mast cells and eosinophilsrelease NET-like structures in response to inflammatory stimuli andreactive oxygen species (von Kockritz-Blickwede et al., Blood 2008111:3070-3080; Yousefi et al., Nature Medicine 2008 14:949-953).

As demonstrated herein, nucleosomes are externalized during ischemicbrain injury. The data presented herein show that mice deficient forDNase-1 are more susceptible to ischemic stroke and that wild-type micetreated with rhDNase-1 are protected from stroke; suggesting aprotective effect of DNase-1 in ischemic brain injury. Indeed, as shownherein, in in vitro flow chambers, DNase-1 was able to removeDNA/platelet complexes from the surface, preventing DNA-mediatedplatelet adhesion and aggregation.

It is shown herein that histones are a potent platelet aggregationagonist and that pharmacological targeting of histone H2A/H4 leads to asignificant protective effect in ischemic stroke, suggesting thathistones contribute to ischemic brain injury during stroke.Pharmacological blocking of histone mediated plateletactivation/aggregation and endothelial dysfunction can thus also help toattenuate ischemic stroke progression.

In conclusion, these data indicate that NETs are an important mediatorof ischemic stroke in mice and that anti-NET compounds, particularlyDNase, could be useful as a therapeutic agent in stroke management. Asdemonstrated herein, removal of excess chromatin in brain vessels couldlimit obstructive events mediated by NETs and preventing localaccumulation of toxic and platelet-activating histones. DNase-1 is areadily available drug that is currently used for treatment of cysticfibrosis to reduce sputum viscosity by digesting DNA released fromneutrophils (Lieberman, JAMA 1968 205:312-3).

Example 4: Anti-NET Compounds in the Treatment of Deep Vein Thrombosis

The role of NETs in deep vein thrombosis (DVT) was also examined usingthe DVT model of flow restriction in the inferior vena cava. Mice wereanesthetized by isoflurane-oxygen mixture and placed in a supineposition. After laparotomy, intestines were exteriorized and warmsterile saline was applied during the whole procedure to prevent drying.After gentle separation from aorta, IVC was ligated over a 30 G needleby a 7.0 polypropylene suture immediately below the renal veins (towardsthe tail) and then the needle was removed. The needle was placed outsidethe vessel so that piercing or any other injury to the IVC wall wascompletely avoided. This procedure allows for standardized flowrestriction without endothelial injury. All visible side branches(usually 1 or 2) were also ligated. After surgery, peritoneum and skinwere closed by monofilament absorbable suture and 6.0 silk,respectively. Mice were euthanized after 6 h or 48 h and thrombideveloped in the IVC below the suture (towards the tail) were taken foranalysis.

All mice were treated with DNaseI (Pulmozyme®) immediately after surgeryand mice with 48 h IVC stenosis received 3 additional injections every12 hours. The dose of every injection was 50 μg i.p. (50 μl ofnon-diluted Pulmozyme) and 10 μg i.v. (in 50 μl of sterile saline) inretro-orbital plexus. Control mice were injected with the same volume ofDNase buffer (8.77 mg/ml sodium chloride and 0.15 mg/ml calcium chloridein sterile water for injections).

In the 6 h IVC stenosis model, half (7 of 14) of the vehicle-treatedmice produced a thrombus whereas in mice that received DNase I, only 1mouse of 10 formed a thrombus (P<0.05 by the chi-square test; FIG. 13C).Sixty two percent (5 of 8) of control mice produced a thrombus 48 hafter flow restriction application. In contrast, only 17% (2 of 12) ofDNase I-treated mice developed a thrombus (P<0.04; FIG. 13F). Weights(FIG. 13A, D) and lengths of thrombi (FIG. 13B, E) were alsosignificantly reduced in DNase I-treated mice in both the 6 h and 48 hIVC stenosis experiments.

Example 5: Anti-NET Compounds in the Treatment of Pulmonary Embolism

To further investigate whether prophylactic treatment with an anti-NETcompound could prevent cardiovascular conditions, mice were administeredDNase prior to pulmonary embolisms being induced. Male C57Bl/6J mice(22-26 g body weight, 7 weeks old) were injected with DNaseI (50 mg) orII (600 U) intraperitoneally. Thirty min later, mice were put to sleepby intraperitoneal injection of avertin (2.5%). Fifty min after theinitial DNase injection, mice received intravenous infusion of DNaseI(10 mg) or DNaseII (120 U) in 50 ml buffer. Control mice were injectedwith the buffer at the same time points.

One hour after initial DNase or buffer injection, mice receivedintravenous infusion of the mixture of 0.8 mg/kg collagen (Nicomed) and60 mg/kg epinephrine (Phoenix) in 100 ml sterile PBS. Mice weremonitored for 1 h. All mice had an episode of breath insufficiency whichproves that the injection of collagen/epinephrine mixture wassuccessful. Data were compared statistically using the Chi-square test.

As shown in FIG. 14, a large proportion of mice which receivedprophylactic doses of DNaseI or DNaseII were able to survive theinjection of collagen/epinephrine while mice receiving the controlinjections experienced 100% mortality.

Example 6: Anti-NET Compounds in the Treatment of TRALI

In order to determine the effect of anti-NET compounds as a treatmentfor TRALI, a mouse model of the 2-hit TRALI model was utilized. BALB/cmale mice (8-10 week-old) were primed with an intraperitoneal injectionof lipopolysaccharide (LPS) (0.1 or 0.5 mg/kg) 24 hours prior tochallenge with anti-H2K^(d)mAb (clone 34-1-2s1, 1 mg/kg) or isotypecontrol injected retro-orbitally. Before each injection mice wereshortly anesthetized by inhalation of isoflurane. To measure the effectof DNaseI treatment on mice experiencing TRALI, mice received intranasalDNaseI (50 μg/mouse, 1 μg/ul) or 50 μl of the buffer-vehicle 10 minutesprior to the antibody injection.

Induction of TRALI by administration of LPS and anti-H2K^(d) mAb causeddamage to the epithelium of the lungs and a dense coat of fibrousmaterial was detectable (FIG. 15B). The fibrous material was absent inlungs from untreated mice (FIG. 15A) and in the lungs of mice with TRALIwhich received DNase 1 treatment (FIG. 15C). The progression of TRALIwas also measured by determining the platelet counts and the proteinconcentration found in bronchial alveolar lavage (BAL) of the mice.Whereas mice suffering from TRALI and receiving control treatments werefound to have decreased platelets (FIG. 16A) and increased BAL proteinlevels, (FIG. 16B), these effects were diminished if the mice receivedintranasal administration of DNaseI (FIGS. 16A-16B).

Example 7: PAD4 and DVT

First described by Brinkmannn et al. in 2004, there is a rapidly growingliterature showing that NETs play an important role in the innate immuneresponse, particularly by their entrapment of extracellular pathogens(Brinkmannn et al. Science 2004). Furthermore, NETs are involved in thepromotion of thrombus formation (Fuchs et al. PNAS 2010) and can inducecoagulation (Massberg et al. Nat. Med. 2010). While little is currentlyknown about the molecular mechanisms involved in the process of NETformation, histone hypercitrullination by the enzyme peptidylargininedeiminase 4 (PAD4) has been demonstrated in the NET formation process(Neeli et al. J Immunol 2008, Wang et al. J Cell Biol 2009). PAD4 is amember of the PAD family of enzymes, which convert protein arginineresidues to citrulline through a deimination reaction. PAD4 is the onlyPAD family member with a nuclear localization signal and therefore canenter the nucleus to modify histones (Nakashima et al. J Biol. Chem.2002) In PAD4^(−/−) mice, a complete loss of NETs formation was seen invitro along with a resulting impairment in innate immune responses tobacterial infection in vivo (Li et al. J Exp. Med. 2010). Therefore, therole of this histone modification in the process of NET formation is ofgreat interest.

Described herein is data indicating PAD4 involvement in several modelsinvolving neutrophils in the lab as seen by immunofluorescence stainingof tissue and isolated neutrophils for citrullinated histone H3, aproduct of PAD4 activity.

Using mouse models of deep vein thrombosis (DVT), we have investigatedthe role of PAD4 and NETs in vivo. As described elsewhere herein, theinventors have previously demonstrated that biomarkers of NETs areabundant in baboon and mouse DVT. Pretreatment of mice with DNaseI,which degrades NETs, greatly reduces the incidence of thrombosis inwild-type mice. Described in this Example herein, is a venous stenosismodel of DVT in mice in which PAD4^(−/−) mice are being studied.Briefly, the inferior vena cava is ligated to induce a 90% restrictionin blood flow. This results in the formation of a thrombus that issimilar in composition to human deep vein thrombi: a platelet-rich whiteportion distal to the ligation site and an erythrocyte-rich red portionproximal to the ligation site, with neutrophils present throughout thethrombus. Neutrophils with citrullinated histones and extracellularH3Cit were identified in these thrombi at 48 hours after ligation (datanot shown).

A lack of NET formation in these mice should result in reduced thrombussize and/or frequency of formation. Since PAD4-deficient mice do notform NETs in vitro, it was hypothesized that NET formation would begreatly impaired in these mice and this would either prevent or delaythrombus formation in these mice. Therefore, the IVC stenosis model ofDVT was examined in PAD4^(−/−) mice with C57Bl/6 mice (wld type) ascontrols. Mice were anesthetized with isoflurane and given buprenorphineas an analgesic prior to beginning surgery. A midline laparotomy wasperformed and the inferior vena cava exposed. Side branches of the IVCbelow the renal veins were completely ligated and the IVC was ligated inthe presence of a 30 g spacer immediately below the renal veins. Theinventors have previously seen that this results in a 90% reduction invessel diameter. The mice were then sutured and monitored over a 48-hourperiod. At 48 hours, mice were anesthetized and 300 microliters of bloodwas collected through the retroorbital sinus. The midline incision wasre-opened and the IVC exposed. Presence or absence of a thrombus wasdetermined by cutting open the IVC between the renal veins and the iliacvein. Thrombi were collected, washed, measured for length and thenimmediately frozen for further analysis (FIG. 18A).

While a majority of wild-type mice (9/10) form a thrombus at 48 hours,the incidence of thrombus formation was greatly reduced in PAD4^(−/−)mice (1/11) at this time point (FIG. 18B). These results indicate thatthese mice can be used to further study NETs involvement in thrombosispathogenesis in vivo, and that PAD4 inhibitors will be beneficial inpreventing deep vein thrombosis in humans.

Inhibitors of PAD4 have been previously described (Luo et al.Biochemistry 2006). They have been studied in vitro with primary humanneutrophils and a human granulocyte precursor cell line (HL-60 cells).Cl-amidine has been shown in vivo in mouse models of arthritis (Williset al. JI 2011) and colitis (Chumanevich et al. Am J PhysiolGastrointest Liver Physiol 2011) to reduce clinical signs in bothconditions. Two of these, Cl-amidine and F-amidine, have recently becomecommercially available through Cayman Chemical (Cl-amidine: Catalognumber 10599, CAS 913723-61-2, F-amidine: Catalog number 10610). Theseinhibitors bind to cysteine 645 in the active site of the enzyme,irreversibly inhibiting enzyme activity. These inhibitors are notselective and also inhibit other PAD family members.

REFERENCES FOR EXAMPLE 7

-   Brinkmannn et al. Science 2004, 303:1532-1535.-   Fuchs et al. PNAS 2010 107:15880-15885.-   Massberg et al. Nat. Med. 2010, 16:887-896.-   Neeli et al. J Immunol 2008, 180:1895-1902.-   Wang et al. J Cell Biol 2009, 184: 205-213.-   Nakashima et al. J Biol. Chem. 2002, 277:49562-49568.-   Li et al. J Exp. Med. 2010, 207:1853-1862.-   Luo et al. Biochemistry 2006, 45:11727-11736.-   Willis et al. Journal of Immunology 2011, 186:4396-4404.-   Chumanevich et al. Am J Physiol Gastrointest Liver Physiol 2011,    300:G929-38.

Example 8: Nets Promote Deep Vein Thrombosis in Mice

Upon activation, neutrophils can release nuclear material known asneutrophil extracellular traps (NETs), which were initially described asa part of antimicrobial defense. Extracellular chromatin was recentlyreported to be prothrombotic in vitro and to accumulate in plasma andthrombi of baboons with experimental deep vein thrombosis (DVT).Described herein is the exploration of the source and role ofextracellular chromatin in DVT using an established murine model of DVTinduced by flow restriction (stenosis) in the inferior vena cava (IVC).It is demonstrated herein that the levels of extracellular DNA increasein plasma after 6 h IVC stenosis, compared with sham-operated mice.Immunohistochemical staining revealed the presence of Gr-1-positiveneutrophils in both red (RBC-rich) and white (platelet-rich) parts ofthrombi. Citrullinated histone H3 (CitH3), an element of NETs'structure, was present only in the red part of thrombi and wasfrequently associated with the Gr-1 antigen. Immunofluorescent stainingof thrombi showed proximity of extracellular CitH3 and von Willebrandfactor (VWF), a platelet adhesion molecule crucial for thrombusdevelopment in this model. Infusion of Deoxyribonuclease 1 (DNase 1)protected mice from DVT after 6 h and also 48 h IVC stenosis. Infusionof an unfractionated mixture of calf thymus histones increased plasmaVWF and promoted DVT early after stenosis application. Extracellularchromatin, likely originating from neutrophils, is a structural part ofa venous thrombus and both the DNA scaffold and histones contribute tothe pathogenesis of DVT in mice. NETs provide new targets for DVTtreatment.

Material and Methods

Mice. Wild-type C57BL/6J (WT) mice were from Jackson Laboratory (BarHarbor, Me., USA). All mice were 7-9-week-old males weighing 22-26 g.

Flow restriction model. The murine model of DVT was performed asdescribed [22]. In brief, mice were anesthetized by isoflurane-oxygen,intestines were exteriorized and the inferior vena cava (IVC) wasdiligently separated from aorta. A suture was placed on the IVC justbelow the renal veins over a spacer (diameter of 0.26 mm) and then thespacer was removed. This procedure has been shown to decrease vascularlumen by about 90% and avoid endothelial injury. All visible IVC sidebranches were also sutured. Thereafter, peritoneum and skin were closed,mice were sacrificed after 1-48 h and thrombi formed in the IVC wereharvested. Sham-operated mice were opened and IVC sutured similarly tothe experimental mice, but the suture was removed immediately afterligation.

Histone infusion. A mixture of all histones isolated from calf thymus(Worthington, Lakewood, N.J., USA) was dissolved in sterile saline andinfused intravenously in mice immediately before IVC stenosisapplication. The dose used (10 mg kg⁻¹) is known to produce a 25%decrease in platelet count 10 min after infusion [6]. The infusedsolution of histone mix was essentially endotoxin-free (<0.025 EU mL⁻¹;measured using the endotoxin detection kit [Lonza, Walkersville, Md.,USA] according to the manufacturer's instructions). Control micereceived infusion of sterile saline. Mice were sacrificed 1 h aftersurgery and thrombus formation was examined.

DNase 1 infusion. DNase 1 (PULMOZYME®, Genentech, San Francisco, Calif.,USA) was diluted in sterile saline and injected immediately aftersurgery (50 μg intraperitoneally and 10 μg intravenously). Inexperiments with 48 h IVC stenosis, injections were repeated three moretimes after every 12 h. Control mice were injected with the PULMOZYME™vehicle buffer (8.77 mg mL⁻¹ sodium chloride and 0.15 mg mL⁻¹ calciumchloride) diluted in sterile saline.

Determination of extracellular DNA in plasma. Blood (100 μL) was drawnfrom the periorbital eye plexus and stabilized with 5 μL of 0.5 M EDTA.Time points for blood drawing were: 24 h before (all mice) and theneither 6, 24 and 48 h after DVT (three mice) or sham surgery (fourmice), or 6 and 48 h after DVT (three mice) or sham surgery (four mice).Plasma was obtained by centrifugation at 2300 g, diluted 50-fold withPBS containing 0.1% BSA, mixed with an equal volume of 1 μM of thefluorescent DNA dye SYTOXGREEN™ (Invitrogen, Carlsbad, Calif., USA) andfluorescence of dye bound to DNA was immediately determined by afluorescence microplate reader (Fluoroskan, Thermo Scientific, Waltham,Mass., USA) as described [4]. Background fluorescence of PBS-plasmamixture (without SYTOXGREEN™) was subtracted from all samples.

Frozen sections. Thrombi with or without the surrounding IVC wall orsham IVC (IVC fragment of 6-8 mm ligated at both ends with bloodremaining inside) were harvested, embedded in Optimal CuttingTemperature (OCT) compound (Sakura Finetek, Torrance, Calif., USA) andthen cryosectioned into 10-μm sections.

Immunohistochemistry. Immunohistochemistry was performed as previouslydescribed [24]. Briefly, anti-mouse Gr-1 antibody (dilution 1:500, cloneRB6-8C5; BD Pharmingen, Franklin Lakes, N.J., USA) and rabbit polyclonal[CitH3] antibody to citrullinated histone H3 (dilution 1:300, citrulline2+8+17; ab5103, Abcam, Cambridge, Mass., USA) were used as firstantibodies. HISTOFINE SIMPLE STAIN MOUSE MAX PO™ for rat (414311F) andrabbit (414351F), respectively, purchased from Nichirei Corporation(Tokyo, Japan), were used as secondary antibodies. Diaminobenzidine(DAB) substrate kit (Vector Laboratories, Burlingame, Calif., USA),containing DAB and DAB-Ni, was used for visualization of staining.Finally, sections were counterstained with Nuclear Fast Red(Sigma-Aldrich, St Louis, Mo., USA). No first antibodies were applied incontrol sections.

Immunofluorescent staining. Sections were incubated with zinc fixativefor 15 min, washed in PBS, and permeabilized with 0.1% Triton X-100,0.1% sodium citrate on ice. After washing and blocking with 3% bovineserum albumin (BSA, Sigma-Aldrich), the sections were incubated for 16 hat 4° C. in 0.3% BSA in PBS with 0.3 μg mL⁻¹ rabbit polyclonalanti-CitH3 (citrulline 2+8+17; ab5103, Abcam), and sheep polyclonalanti-VWF (1:50 dilution of IgG fraction; ab11713, Abcam). Forlongitudinal sections of isolated thrombi, anti-CitH3 Ab was appliedovernight and anti-VWF Ab for 90 min. After washing, sections wereincubated with the following ALEXA FLUOR™-conjugated secondaryantibodies (all from Invitrogen) in 0.3% BSA in PBS: ALEXA FLUOR 488donkey anti-rabbit IgG and ALEXA FLUOR 568 donkey anti-sheep IgG (2 μgmL⁻¹ for all Abs) for 2 h at room temperature. DNA was labeled with 1 μgmL⁻¹ Hoechst 33342 (Invitrogen). Fluorescent images for cross-sectionswere acquired using an AXIOVERT™ 200 inverted widefield fluorescencemicroscope (Zeiss, Thornwood, N.Y., USA) in conjunction with a ZeissAXIOCAM MRM™ monochromatic CCD camera. Mosaic reconstruction of entirecross-sections was performed with MOSAICJ™ [25] for IMAGEJ™ software(National Institutes of Health, Bethesda, Md., USA; available on theworld wide web at http://rsbrreb/nih/gov/iji) and consists of two to sixfields of view per image shown. Images for longitudinal sections wereobtained by a widefield fluorescence microscope using an AXIOPLAN™microscope (Zeiss) with color HRc Zeiss camera. Images were analyzedwith AXIOVISION™ software (Zeiss).

Plasma VWF measurement. The assay was performed as described [22]. Thelevel of VWF in pooled plasma of 20 C57BL/6J WT mice was used as areference standard.

Statistics. Results obtained on the same animal at different time points(difference in plasma DNA levels between baseline and 6 h and plasma VWFcontent) were compared using paired Student's t-test. Plasma DNA levelsin mice with IVC stenosis and sham-operated animals were compared byMann-Whitney test. Difference in thrombi prevalence between differentgroups of mice was compared using a contingency table and the chi-squaretest. Differences were considered significant at P<0.05.

Results

Flow Restriction in the IVC Promotes Plasma DNA Accumulation

IVC stenosis was performed in WT mice. Blood was drawn 24 h before and6, 24 and 48 h after surgery and plasma DNA levels were measured. Plasmaof non-operated mice contained 284±18.9 ng mL⁻¹ DNA. Stenosis of the IVCled to an increase in plasma DNA levels 6 h post surgery (FIG. 21) to515±34.7 ng mL⁻¹ (P<0.003 vs. baseline and P<0.03 vs. sham-operatedmice). This effect did not result from the surgical procedure becausesham-operated mice had plasma DNA levels similar to non-operated animals(326±47.4 ng mL⁻¹, P=0.9). The concentration of DNA in plasma 48 h aftersurgery returned to baseline and was not different in DVT- andsham-operated mice. Appearance of DNA in plasma 6 h after stenosisapplication, when about half of the mice form visible thrombi (asdescribed in the text), indicates that chromatin release occurs early inthe thrombotic process.

Venous thrombi contain CitH3 located predominantly in the red part ofthe thrombus.

Besides neutrophils, other cell types can release chromatin [8-10].Histone citrullination by PAD4 occurs in activated granulocytes and isnecessary for the formation and release of NETs [14-16,26]. To assessthe presence of NETs in venous thrombi, thrombi developed in mice after48 h IVC stenosis were stained for CitH3. The white part of the thrombus(the platelet-rich part remote from the suture) contained numerousGr-1-positive cells but was essentially devoid of CitH3 (data notshown). Gr-1 is an antigen present on polymorphonuclear leukocytes andalso on plasmacytoid dendritic cells and a small subset of monocytes(reviewed in [27]). The large red part of the thrombus (the RBC-richpart proximal to the suture) contained loci heavily stained for eitherGr-1 only (or both Gr-1 and CitH3 with substantial overlap of the twoantigens (data not shown). Some Gr-1-positive cells formed extracellularfiber-like structures that strongly stained for CitH3, probablyrepresenting NETs.

VWF can play a role in venous thrombosis initiation [22]. As describedabove herein, in baboon DVT thrombi, extracellular DNA was shown toco-localize with VWF. Immunofluorescent staining for VWF in murinevenous thrombi was often associated with extracellular CitH3 staining inthe red part of thrombi (data not shown). Multiple CitH3-positive cellswere also revealed by the immunofluorescent staining in the core of thered part of thrombi, confirming the immunohistochemical data describedabove herein (data not shown). No substantial CitH3 staining wasobserved in the white part of thrombi, IVC wall, sham-operated IVC, andin control sections stained without first antibody. Thus, histonespresent in murine IVC thrombi are likely to originate from neutrophilsreleasing NETs predominantly in the red part of thrombi. In mice,similar to baboon, thrombi extracellular chromatin frequentlyco-distributes with VWF.

Histones increase plasma VWF levels and promote DVT.

Extracellular histones are cytotoxic to endothelial cells and activateplatelets in vitro; histone infusion at a high dose of 75 mg kg⁻¹ islethal to mice [4,28]. Therefore a lower dose (10 mg kg⁻¹) of histonemix, a dose that produces only mild thrombocytopenia [6], was used totest whether it renders mice more prone to venous thrombosis. Only twoof 14 vehicle-treated mice (14%) produced thrombi after 1 h IVC stenosis(FIGS. 22A-22C), while five of nine mice that received histones prior tothe surgery (55%) developed a thrombus (P<0.04).

Histones have been shown to cause Ca²⁺ influx in different cell types[6,29,30]. Increase in intracellular Ca²⁺ level triggers VWF secretionfrom endothelial cells and platelets, with some of the VWF remainingassociated with the plasma membrane of these cells [31]. Plateletrecruitment mediated by VWF is a key step in the initiation of venousthrombosis in this model of DVT [22]. Therefore stimulation of VWFsecretion could be one of the mechanisms responsible for theprothrombotic effect of histones. Indeed, histone infusion increasedplasma VWF levels compared with baseline (Table 1). In contrast, VWFplasma levels in vehicle-treated mice remained unchanged. Thus, histoneinfusion increases plasma concentration of VWF, which could contributeto the effect of histones on DVT in the flow restriction setting.

DNase 1 Infusion Protects Mice from DVT

Based on the presence of histones, DNA and NET-like structures in deepvein thrombi in baboons and mice (as described herein) and on theability of DNase 1 to disassemble NET-induced thrombi in a flow chamber,it was hypothesized that DNase 1 might prevent DVT in vivo. To test thispossibility, DNase 1 was infused in mice immediately after surgery andthrombosis examined after 6 or 48 h of IVC stenosis (in 48-h DVTexperiments, infusions were repeated every 12 h). In the 6-h model, half(seven of 14) of the vehicle-treated mice produced a thrombus (FIGS.13A-13F), whereas in mice that received DNase 1, only one mouse of 10formed a thrombus (P<0.05). In the 48-h IVC stenosis model, mice treatedwith control buffer developed a thrombus in 63% of cases (five ofeight), whereas thrombus prevalence in mice treated with DNase 1 was 17%(two of 12, P<0.04). These data suggest that extracellular chromatin mayplay a role in flow restriction-induced thrombosis and DNase 1 infusionis protective against thrombosis in this model.

Several cell types have been shown to release extracellular chromatinupon activation. The role of the nuclear material originating fromneutrophils, NETs, in antimicrobial defense has been convincinglydemonstrated [2]. Described herein is data implicating extracellularchromatine in thrombosis because NETs can form a scaffold able torecruit both platelets and RBCs in vitro. Perfusion of blood over NETsin a flow chamber results in the formation of a red thrombus, which wasentirely NET-dependent as DNase 1, which destroys NETs, preventedrecruitment of both cell types. This suggested a mechanistic linkbetween NETs and DVT because (i) recruitment of platelets is one of theearly events pivotal for thrombus initiation in mice and (ii) DVTthrombi are rich in RBCs. Thrombi developed in the murine flowrestriction model of DVT also consist of a large RBC-rich red part and asmaller platelet-rich white part with both parts containing fibrin.Thus, thrombi formed in this murine model share close morphologicalsimilarity to human DVT thrombi, which also include white and red parts.

Thrombi obtained in an experimental DVT model in baboons have been shownherein to contain extracellular DNA, H3 and DNA/H2A/H2B complex. It isfurther demonstrated herein that histone H3 is also abundantly presentin murine DVT thrombi. Histone H3 was citrullinated, indicating that itis likely to have originated from neutrophils forming NETs. PAD4, theenzyme responsible for arginine conversion to citrulline, is abundantlyexpressed in granulocytes [15,16]. Staining for Gr-1, a neutrophilmarker, revealed neutrophil presence in both red and white parts of themurine venous thrombi. Neutrophil-specific staining in the red part ofthrombi was frequently associated with CitH3, with CitH3 being eitherconfined to nuclei or localized extracellularly. Without wishing to bebound by theory, this suggests that here neutrophils are at differentstages of NETosis. Interestingly, little CitH3-positive staining wasobserved in the white part of thrombi despite abundant presence ofGr-1-positive cells. As NETosis is an irreversible process, one mayspeculate that neutrophils in the white part have spent less time in thethrombus compared with the red part neutrophils. This may suggest thatthe white part, originally adjacent to the stenosis site where thrombusgrowth begins, has a role in recruiting neutrophils from the surroundingblood. This is likely to be through binding to the activated platelets.Later on, these neutrophils may also become activated and form NETs,which in turn would contribute to recruitment of RBCs and the formationof red thrombus.

The model described herein corroborates the reported ability ofstimulated platelets to bind neutrophils and induce formation andrelease of NETs [3,5]. In addition, flow restriction may create hypoxicconditions in the vessel wall and cells buried inside a thrombus areexposed to even more severe hypoxia due to isolation from the bloodstream. Hypoxia potentiates the release of ROS [32]. Besidesneutrophils, platelets also can generate ROS, such as superoxide [33].It has been shown that ROS not only directly contribute to thrombosis[34] but also can trigger formation of NETs [13,35]. Therefore, theadherent neutrophils exposed to two major triggers of NETs production,activated platelets and ROS, release extracellular chromatin, which thencontributes to further thrombus development.

At the early stages of flow restriction, massive recruitment of bothplatelets and leukocytes to the endothelium occurs simultaneously [22].It has recently been reported that co-culture of neutrophils withactivated endothelial cells can also induce NET formation, which in turnpromotes endothelial damage [36]. As the activation state of theendothelium is critical for DVT initiation [22], it is possible thatNETs-endothelial interactions could be involved in thrombus initiation.This fits with the observation made herein that NETs biomarkersaccumulate in plasma within hours after flow restriction induction andthat histones cause an increase in plasma VWF levels, probably from theactivated endothelium.

As NETs are generated during the early stage of thrombus initiation andare also abundantly present in mature thrombi, it would be reasonable tohypothesize that NETs degradation might affect thrombosis. Here,infusion of DNase 1 protected mice from flow restriction-induced DVTregardless of the length of stenosis (6 or 48 h, FIG. 13A-13F). Withoutwishing to be bound by theory, as no visible thrombus was detected inmost DNase 1-treated mice, DNase 1 apparently cleaves NETs early,disrupting the pathways of cellular activation and preventing thecascade of events leading to thrombosis. Without wishing to be bound bytheory, the anti-thrombotic effect of DNase 1 is likely to be mediatedby removal of NETs generated locally at the site of stenosis, similar tocleavage of endothelium-bound VWF by ADAMTS13. Similar to ADAMTS13,DNase 1 infusion may not reduce the amount of circulating DNA but ratheraffect its size and local concentration.

It is known that blood coagulation contributes to DVT andhypercoagulable states are considered risk factors for the disease[19-21]. The murine DVT model used herein recapitulates this feature ofhuman DVT because fibrin could be detected throughout the thrombus [22].Although anticoagulants have not been tested in this model, aprocoagulant state, such as in mice with high plasma levels of solubleP-selectin [38], is associated with increased DVT induced by stasis inthe IVC [39].

As described herein, strings of extracellular CitH3 frequentlyco-localized with VWF in the thrombi produced by IVC stenosis. Thisfinding corroborates in vitro observations that histone [40] and NETsbind VWF [4]. Secretion of VWF from Weibel-Palade bodies (WPBs) to thesurface of endothelial cells appears required for the development of DVTin mice [22].

Interestingly, VWF expression is downregulated in the venous valvularsinus, which experiences stasis and hypoxia, likely to maintain athromboresistant phenotype at this thrombosis-susceptible site [41]. Inthrombi, VWF may originate not only from endothelium but also fromplatelets, in which it is stored in alpha-granules. VWF and NETs mayform a mutually supportive network that contributes to VWF A1 domainactivation [31] and to growth and stabilization of a venous thrombus.

Histones also are likely to participate in the process of thrombusinitiation. Infusion of histone mix facilitated thrombosis in mice(FIGS. 22A-22C). This may result from the observed deleterious effect ofhistones on endothelium in vitro [28], which at a lower dose in vivomight activate endothelium and stimulate the release of VWF as observedherein (Table 1). Another prothrombotic effect of histones could resultfrom activation of platelets [4,6]. Activated platelets can stimulateNETs production [3,5] and promote release of WPBs [42], leading tofurther recruitment of platelets and leukocytes. In either case,histones would contribute to the process of thrombus initiation andpropagation.

In conclusion, it is demonstrated herein that NETs have an importantfunctional role of NETs in DVT induced by flow restriction, therebyproviding targets for treatment of DVT

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TABLE 1 Histone infusion elevates plasma VWF levels. Blood was drawn 24h before and 1 h after infusion of saline or histone mix (10 mg kg⁻¹). Pvalue was calculated by the paired t-test. Saline- injected mice, n = 8;histone-injected mice, n = 6 Saline Histone mix Baseline 1 h afterinfusion Baseline 1 h after infusion 83 ± 2.7% 78 ± 5.2% 78 ± 4.2% 94 ±6.7% — P = 0.43 — P <0.003

Example 9: Extracellular DNA Traps are Formed During TRALI

Transfusion-related acute lung injury (TRALI) is the leading cause oftransfusion-related death. The biological processes contributing toTRALI are poorly understood. All blood products can cause TRALI, and nospecific treatment is available. A “two-event model” has been proposedas the trigger. The first event may include surgery, trauma orinfection; the second involves the transfusion of anti-leukocyteantibodies or bioactive lipids within the blood product. Together, theseevents induce neutrophil activation in the lungs, causing endothelialdamage and capillary leakage. Neutrophils, in response to pathogens orunder stress, can release their chromatin coated with granule contents,thus forming neutrophil extracellular traps (NETs). Although protectiveagainst infection, these NETs are injurious to tissue. It isdemonstrated herein that NET biomarkers are present in TRALI patients'blood and that NETs are produced in vitro by primed human neutrophilswhen challenged with anti-HNA-3a antibodies previously implicated inTRALI. NETs are found in alveoli of mice experiencing antibody-mediatedTRALI. DNase 1 inhalation prevents their alveolar accumulation andimproves arterial oxygen saturation even when administered 90 minutesafter TRALI onset. Accordingly, NETs can be targeted to prevent or treatTRALI.

TRALI is a rare but serious complication of blood transfusion thatoccurs within 6 hours of transfusion and is characterized by hypoxemic,respiratory distress and pulmonary infiltrates.¹ Over the past yearsprevention measures have resulted in a significant reduction in cases.However, TRALI is still the leading cause of transfusion-relatedmortality and its prevalence is likely underestimated; one studysuggested that over 2% of cardiac surgery patients are affected.² Onlysupportive treatment is available to the patient, including mechanicalventilation and oxygen supplementation. Many of the severe cases havebeen linked to the presence of anti-neutrophil antibodies in thetransfused product.^(3,4) These antibodies bind to the recipients'neutrophils, activate them and induce sequestration in the pulmonarycapillaries, resulting in tissue injury.⁵ Activated neutrophils canrelease neutrophil extracellular traps (NETs)⁶ that are composed of DNAfibers decorated with histones and antimicrobial proteins⁷ originallycontained in the neutrophil granules. The structure and the compositionof NETs allow them to trap and prevent the spread of pathogens and alsoto kill Gram-negative and Gram-positive bacteria, as well as yeast.⁶ NETformation follows a specific pattern characterized by histonehypercitrullination,⁸ chromatin decondensation, dissolution of thegranular and nuclear membranes and cytolysis.⁹ Despite NETs' beneficialantimicrobial function,^(6,10) their formation at the wrong time, in thewrong place or in the wrong amount can have a negative effect on thehost. NETs and their components can be injurious to tissue¹¹⁻¹³ and theyhave been shown to contribute to the pathology of several inflammatorydiseases.¹²⁻¹⁷

The purpose of the experiments described in this Example was todetermine whether NETs are formed in patients with TRALI and contributeto TRALI in a mouse model. Antibodies implicated in severe TRALI anddirected against the human neutrophil alloantigen-3a (HNA-3a) have beenidentified and shown to bind to choline-like transporter 2 (CLT-2) onthe recipients' neutrophils.^(18,19) It was evaluated herein whether theantibody enhances NET formation in vitro in human neutrophils expressingHNA-3a. Also investigated herein was whether NETs were formed in thelungs of mice with TRALI.

Methods

Human Samples.

Blood samples from five patients with TRALI, three blood donors whoseplasma caused TRALI and eleven healthy control subjects were included inthe study. TRALI was diagnosed in patients according to theinternational consensus definition.¹ Studies involving human subjectswere approved by the Institutional Review Boards of the Immune DiseaseInstitute and the Blood Center of Wisconsin. The investigation conformsto the principles outlined in the Declaration of Helsinki.

Experimental Mice.

Experiments were performed using 8-10 week old BALB/c male micepurchased from the Jackson Laboratory. All mice were housed in theanimal facility at the Immune Disease Institute. Experimental proceduresperformed on the mice were approved by the Animal Care and Use Committeeof the Immune Disease Institute.

Two-Event TRALI Model.

The model was adapted from Looney et al. (2009).²⁰ Male BALB/c mice(8-10 week old) were primed with an i.p. injection of LPS (0.1 or 0.5mg/kg, as indicated in the text) 24 hours prior to challenge withanti-H-2K^(d) mAb (clone 34-1-2s, 1 mg/kg) or isotype control injectedretro-orbitally. In experiments involving DNase 1 treatment, mice thatwere injected with both LPS and the anti-H-2K^(d) mAb received i.n.DNase 1 (PULMOZYME®, Genentech, 50 μg/mouse, 1 μg/ul) 10 minutes beforeor 90 minutes after antibody injection. Control TRALI mice were injectedwith 50 μl of the DNase 1 vehicle-buffer in DNase 1 experiments. Bloodcollection, lung harvesting and arterial oxygen saturation measurementswere all performed 2 hours after antibody injection. No event ofTRALI-related death was recorded under these conditions in any of thetreated mice.

Body Temperature Measurements.

Rectal temperatures were measured as an indicator of shock-likecondition 2 hours post anti-H-2K^(d) infusion using a rectal temperatureprobe (MOUSEOX PLUS® system, STARR Life Sciences) connected to aPOWERLAB™ data acquisition system (ADInstruments, USA).

Antibody Preparation.

Anti-HNA-3a antibodies from blood donors whose plasma induced TRALI inpatients and control IgG from a control donor were purified using aprotein G-Sepharose column (GE Healthcare). F(ab′)₂ fragments weregenerated from purified anti-HNA-3a antibody using the F(ab′)₂Preparation Kit (Pierce). Quality of the obtained F(ab′)₂ was verifiedafter electrophoresis on SDS-PAGE gel and silver stain. Anti-H-2K^(d)antibodies were purified from the hybridoma 34-1-2s with a proteinA-Sepharose column (GE Healthcare). The antibody preparation was thendialyzed and tested by electrophoresis on SDS-PAGE gel followed bysilver stain, dot-blot and flow cytometry.

Blood Counts.

Mouse peripheral blood counts were measured from EDTA(ethylenediaminetetraacetic acid)-anti-coagulated blood with a HEMAVET™950 veterinary hematology analyzer (Drew Scientific). This blood counteruses two different currents to analyze the cells. A low voltage currentdetermines the volume and the number of cells (Coulter Impedanceprinciple) and a high voltage current that passes through the cellsgenerates information on their internal structure such as nucleus,fluid, granules and vacuoles.

Bronchoalveolar Lavages (BAL).

Mice were sacrificed and intubated with a 24-gauge cannula after theirtracheas were surgically isolated. The lungs were flushed three timeswith 0.8 ml of sterile PBS. BAL fluids were centrifuged for 5 minutes at800 g. Protein concentration was quantified in the supernatant using thebicinchoninic acid method.

Measurements of Blood Arterial Oxygen Saturation.

Blood arterial oxygen saturation was recorded 2 hours afteranti-H-2K^(d) antibody injection, using a small rodent oxymeter sensor(MOUSE^(OX)™, STARR Life Sciences) mounted on the collar of each testedmouse. The 2 hour endpoint was used as per the model published by Looneyand colleagues in 2009.²⁰⁻²²

Human Neutrophils.

Human neutrophils were isolated from blood by dextran sedimentationfollowed by Ficoll-Hypaque gradient centrifugation. Briefly, 10 ml ofblood containing EDTA were diluted in 10 ml of 3% Dextran and sedimentedfor 20 minutes. The leukocyte-rich, erythrocyte-poor upper fraction wasthen centrifuged at 500 g for 10 minutes to pellet the leukocytes.Leukocytes were layered with Ficoll-Hypaque and separated undercentrifugation for 30 minutes at 400 g. Hypotonic red blood cell lysiswas then performed to eliminate the residual erythrocytes and theneutrophils were washed in PBS and resuspended in RPMI containing 10 mMHEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid). Neutrophilpurity was routinely >98% as assessed by differential cell count aftermodified Wright-Giemsa staining. Blood samples from different blooddonors were selected for the ability of their neutrophils to agglutinatein response to plasma containing anti-HNA-3a antibodies.

Granulocyte Agglutination Test.

The granulocyte agglutination test (GAT) was performed as previouslydescribed.¹¹ The agglutination was observed microscopically, and thedegree of agglutination was graded from − (weak) to 3+ (strong).

Net Quantification.

The protocol was adapted from the method of Kessenbrock andcolleagues.¹⁴ HNA-3a-positive neutrophils were allowed to adhere on a96-well plastic plate for 30 minutes at 37° C. The neutrophils were thenprimed with 5 ng/ml TNF-α and incubated with 33 nM (equivalent of 5μg/ml) of control IgG purified from healthy volunteer blood (ControlAb), anti-HNA-3a whole IgG purified from two blood donors whose plasmainduced TRALI (Ab1 or Ab2), anti-CD11a IgG (anti CD11a Ab), F(ab′)2fragments (equivalent to 3.66 μg/ml) prepared from a purifiedanti-HNA-3a IgG preparation from a blood donor whose plasma inducedTRALI (F(ab′)₂), or with 25 nM PMA (positive control). After a 180minute incubation, cells were fixed with 2% PFA and the DNA was stainedusing Hoechst 33342. The released DNA was visualized by fluorescencemicroscopy and percentage of cells making NETs was quantified from 7 to9 non-overlapping fields in 3 wells for each treatment.

Extracellular DNA and Nucleosome Quantification in Plasma.

Plasma DNA and extracellular nucleosomes were quantified according tomanufacturer instructions using commercially available quantificationkits (from Invitrogen and Roche, respectively).

Myeloperoxidase (MPO) Activity Determination.

Mouse lungs were weighed, blended in 50 mmol/L potassium phosphatebuffer, centrifuged, resuspended, and sonicated in potassium phosphatebuffer supplemented with 50 mmol/L hexadecyltrimethylammonium bromide.After centrifugation of the cell lysates, MPO activity was assessed inthe supernatant by adding tetramethylbenzidine and absorbance reading at450 nm after stopping the reaction with sulfuric acid. For MPOquantification in mouse plasma, plasma was diluted in potassiumphosphate buffer supplemented with 50 mmol/L hexadecyltrimethylammoniumbromide and MPO activity was measured as previously described for lungtissue. MPO was quantified in human samples using a commerciallyavailable ELISA activity assay kit (Invitrogen) according tomanufacturer instructions.

Immunohistology.

Mouse lungs were fixed by intratracheal infusion of zinc fixative, thenexcised and bathed in zinc fixative for 2 hours at 4° C. Intratrachealinflation of the lung allowed us to conserve the three-dimensionalalveolar structure. The lungs were then washed in PBS, embedded andcryosectioned into 10 μm or 50 μm sections for widefield and multiphotonfluorescence microscopy, respectively. Sections were incubated with zincfixative for 15 minutes, washed in PBS, and permeabilized (0.1% TritonX-100, 0.1% sodium citrate) on ice. After washing and blocking (3% BSA),the sections were incubated for 2 to 16 hours at 4° C. in 0.3% BSA with1 μg/ml anti-mouse Gr-1 (Ly-6G/C, clone RB6-8C5, BD Biosciences), 0.3μg/ml rabbit anti-histone H3Cit (citrulline 2, 8, 17) (ab5103, Abcam),or sheep anti-mouse VWF (1:50 dilution of IgG fraction, ab11713, Abcam).Sections were then washed and incubated with the followingAlexa-conjugated secondary antibodies (Invitrogen) in 0.3% BSA: Alexa555-goat anti-rat IgG (2 μg/ml), Alexa 488-donkey ant-rabbit IgG (1.5μg/ml), or Alexa 568-donkey anti-sheep IgG (2 μg/ml) for 2 to 4 hours atroom temperature. DNA was labeled with 1 μg/ml Hoechst 33342(Invitrogen) prior to mounting. Fluorescent images were acquired using aZeiss Axiovert 200 inverted fluorescence microscope in conjunction witha Zeiss AXIOCAM MRM™ monochromatic CCD camera and analyzed withAXIOVISION™ software (Zeiss) or using a Zeiss AXIOPLAN™ uprightfluorescence microscope in conjunction with a Hamamatsu CCD Cameracoupled to an image intensifier (videoscope), and analyzed withSLIDEBOOK™ software (Intelligent Imaging Innovations).

Multiphoton Fluorescence Microscopy.

A custom-built ULTIMA™ system from Prairie Technologies was used. Themicroscope was equipped with a water immersion objective (20×/0.95numeric aperture), a laser scanning microscopy device which incorporatesa computer with beam-conditioning equipment, and a scanhead connected toan Olympus IX50 microscope stand. For multiphoton excitation andsecond-harmonic generation, a MaiTai Ti:sapphire laser (Spectra-Physics)was tuned to 1000 nm to balance excitations of various fluorescentprobes used. Emitted light and second-harmonic signals were detectedthrough 455/70-nm, 525/50-nm and 590/50-nm band-pass filters withnon-descanned detectors for the generation of three-color images.Sequences of image stacks were transformed into volume-rendered movieswith VOLOCITY™ (Improvision) or IMARIS™ (Bitplane Scientific Solutions)softwares. Only 50 μm-thick lung tissue cryosections were analyzed.

Transmission Electron Microscopy.

Mouse lungs were fixed by intratracheal infusion of 1.5%formaldehyde/1.5% glutaraldehyde in sodium cacodylate buffer (pH 7.35),excised and bathed in the same solution for 2 hours at 4° C. The fixedlungs were sliced into 0.5 mm sections; the sections were washedextensively in distilled water, frozen on a liquid-helium cooled copperblock, freeze-dried at −80° C., and coated with 2 nm of platinum at a45° angle with rotation and 10 nm of carbon without rotation. Tissue wasremoved from the metal casting using bleach (Austin's A-1-5.25% sodiumhypochlorite), washed and picked up on formvar-coated 200 mesh coppergrids. Grids were photographed in a JEOL-1200 EX electron microscope at100 kV.

FcγRIIa Blockade.

FcγRIIa (CD32)-binding sites on human neutrophils were blocked byincubation for 15 minutes with the IV.3 antibody25 prior to TNF-a andanti-HNA-3a antibody treatment. The IV.3 antibody was replaced by PBS(vehicle) for control treatment.

Statistical Analyses.

Statistical significance for nonparametric distributions (mouse andhuman samples) was assessed with the two-tailed Mann-Whitney test fortwo groups. The one-way ANOVA test with Bonferroni post-test was usedfor in vitro studies (NET quantifications). GRAPHPAD PRISM™ 4.0 softwarewas used for all analyses. Differences were considered statisticallysignificant at P<0.05. In the figures, significant differences wereillustrated with asterisks (* P<0.05; ** P<0.01; *** P<0.005.

Results

Anti-Neutrophil Antibody Linked to TRALI Promotes NET Formation In Vitro

Because anti-HNA-3a antibodies cause the most severe TRALI,^(3,18,19)whether such antibodies could promote NET formation by human neutrophilswas tested. IgG were purified from two blood donors with anti-HNA-3aantibodies whose plasma caused a TRALI reaction in patients and controlIgG were purified from a control donor's plasma. Neutrophils positivefor the HNA-3a antigen were isolated from healthy volunteers' blood andprimed with 5 ng/ml of tumor necrosis factor-α (TNF-α, a LPS-inducedcytokine). The neutrophils were then incubated with 5 μg/ml of eitheranti-HNA-3a IgG or control IgG for 180 minutes and compared to theeffect of phorbol 12-myristate 13-acetate (PMA) to quantify theircapacity to induce NETs. Using fluorescence microscopy, it was observedthat TNF-α-primed neutrophils formed significantly more NETs uponincubation with the two sources of anti-HNA-3a antibodies (Ab1 and Ab2)compared to neutrophils incubated with control IgG (FIGS. 23A and 23B).Moreover, a majority of the neutrophils treated with the anti-HNA-3aantibody lost their lobulated nucleus morphology. This delobulation wasassociated with a larger nucleus area (FIG. 23C), which is a typicalearly event in the formation of NETs.²³

Because FcγR engagement has been shown to be linked to robust ROSgeneration,²⁴ and ROS generation promotes NET formation,⁹ it washypothesized that FcγR engagement by anti-neutrophil antibodies couldpromote NETosis. To test this hypothesis, F(ab′)₂ fragments wereprepared from anti-HNA-3a IgG purified from a blood donor whose plasmainduced TRALI (Ab1). The incubation of anti-HNA-3a F(ab′)₂ fragmentswith TNF-α-primed neutrophils did not induce NETs generation to agreater extent than TNF-α alone (FIG. 23C). As a second approach, theFcγRIIa (CD32)-binding sites on human neutrophils were blocked with theIV.3 antibody²⁵ prior to TNF-α and anti-HNA-3a antibody treatment. Theincrease in NETosis was no longer observed after anti-HNA-3a antibody(Ab1, Ab2) stimulation (FIG. 24). Together these results indicate thatanti-HNA-3a antibodies trigger neutrophil activation, ultimately leadingto NET generation likely through a FcγRIIa-dependent process.

NET Biomarkers can be Detected in the Bloodstream of Patients with TRALI

In order to determine whether NETs are formed in humans during TRALI,the presence of NET degradation products was assessed in the blood ofpatients with documented TRALI. For that purpose, serum samples from 6healthy volunteers and from 5 patients who developed pulmonaryinsufficiency within 1-6 hours of transfusion and were diagnosed withTRALI (Table 2)¹ were blindly screened. A significant increase incirculating DNA and nucleosome levels was detected in the TRALI patientscompared to the healthy subjects (Table 5). Based on the increase inconcentration of these markers, samples that originated from TRALIpatients versus control individuals were correctly identified. Blooddonors that induced TRALI (Table 3) did not contain excessive DNA ornucleosomes when compared to healthy control subjects, indicating thatNETs formed in the recipients after transfusion (Table 4). Even thoughthe increase in circulating levels of DNA and nucleosomes cannot beexclusively attributed to NET formation, the tendency for serummyeloperoxidase to increase in TRALI patients likely reflects neutrophilactivation.

Antibody-Mediated TRALI Causes NET Formation in the Lungs of Mice

A previously established in vivo model of antibody-induced TRALI in micewas next used.^(20,21,26) In this two-event model, BALB/c mice areinjected i.p. with a low-dose of LPS (0.1-0.5 mg/kg) and 24 hours laterare infused with an anti-MHC class I monoclonal antibody(anti-H-2K^(d)). Two hours later, arterial blood oxygen saturation wasmeasured to document lung function. The mice were then sacrificed andlung injury markers²⁶ quantified (FIG. 25A-25D, data not shown). Asobserved in blood from TRALI patients, a significant increase (1.3 to3.6 fold) in the concentration of NET biomarkers such as DNA,nucleosomes, and myeloperoxidase was detected in the plasma of mice withTRALI compared to mice that were challenged only with LPS (FIGS.26A-26C). Besides endothelial cells, monocytes and platelets,^(20,26,27)neutrophils are essential in the pathophysiology of TRALI.^(3,26) Theirrecruitment to lung tissue combined with their activation induced byantibody transfusion²⁰ led to the hypothesis that NETs are formed in thelungs of mice during TRALI. Transmission electron microscopy (TEM) wasused to address this question (FIGS. 27A-27D). This technique allowedthe detection of a fibrous mesh coating the alveoli within the airspacein the TRALI group (FIGS. 27B and 27D), but such a mesh was not detectedin control mice (FIG. 27A). Since fibrin and DNA cannot be distinguishedby EM²⁸ and because DNase 1 cleaves DNA but not fibrin,²⁹ LPS-primedmice were treated with intranasal DNase 1 administration 10 minutesprior to antibody injection. Two hours after anti-H-2K^(d) mAbchallenge, no fibrous material could be detected in the DNase 1-treatedalveoli (FIG. 27C); thus DNA was the basis of the fibrous mesh in thealveoli of mice with TRALI. These results are supported by widefieldfluorescence microscopy observation of more DNA fibers withmorphological NET characteristics in mice with TRALI, (data not shown)by visualization of diffuse DNA structures of irregular shape and DNAstreaks next to alveoli. The fluorescence microscopy also showed thatthis DNA likely originated from neutrophils or a subset of monocytes(Gr-1 positive). Histone citrullination catalyzed by PAD4(peptidylarginine deiminase 4), an enzyme prominently found ingranulocytes³⁰ has been shown to be a crucial step for neutrophilchromatin decondensation and in NET formation.⁸ Multiphoton analysis offixed lung tissue allowed the observation of areas with robustcitrullinated histone H3 staining colocalizing with DNA streaks in thealveoli outside blood vessels in mice with TRALI. Thus the extracellularDNA was most likely of neutrophil origin. In the model described herein,depletion of Gr-1 positive cells prevented the appearance ofhypercitrullinated histone H3 formation in the lungs of the micesubjected to TRALI whereas platelet depletion did not preclude thepresence of these NET biomarkers in the lungs (data not shown). Therobust staining for citrullinated histone H3 observed in mice with TRALIwas rarely present in LPS-only-treated mice and could not be detected bywidefield fluorescence microscopy in control lungs (data not shown).

NET Disruption in Alveoli During TRALI can Improve Blood Oxygenation inMice

Because intranasal DNase 1 treatment of the mice prior to anti-H-2K^(d)mAb infusion can prevent NET deposition in the lungs, whether it couldimprove lung function in mice with TRALI was examined. Mice subjected toTRALI present transient hypoxia as shown by down spikes in bloodarterial oxygenation saturation (saturation <90%) (data not shown), aphenomenon not observed in control healthy mice (data not shown). Thesedown spikes were significantly attenuated in the mice pretreated withDNase 1 (data not shown) compared to the vehicle buffer-pretreated mice.A general effect of DNase 1 on blood oxygenation can be measured and isdepicted as an increase of the mean arterial oxygen saturation recorded(overall oxygen saturation stability, FIG. 28A) accompanied by anincrease in the minimum arterial oxygen saturation measured during the20 minute-recording time (hypoxia episode intensity, FIG. 28B) whencompared to healthy mice. These results showed that DNase 1 given i.n.as a pretreatment (Pre) but also given i.n. as a treatment 90 minutesafter the onset of TRALI (Post) succeeded in correcting the defect inblood oxygenation observed during TRALI. Body temperatures were alsomonitored as a measure of the shock-like condition induced by theLPS/anti-H-2K^(d) mAb infusion. While the mice with TRALI were showingsigns of hypothermia 2 hours after mAb infusion (FIG. 28C), rectaltemperatures were normal in mice subjected to TRALI and that receivedDNase 1 either as prophylaxis or treatment. These results support thehypothesis that NETs are indeed formed in TRALI lungs and are involvedin the pathophysiology of TRALI.

Discussion

NETs are important in anti-bacterial defense,^(6,7) but several studieshave shown that NETs³¹ and their components, such as histones,^(12,32)elastase¹¹ or pentraxin-3¹³ are injurious to tissues. Histones, at ahigh dose, can even induce death when infused intravenously inmice.^(12,16) In addition, NETs activate blood coagulation³³ andplatelets.^(29,34) Histone infusion causes rapid thrombocytopenia inmice³⁴ and mild thrombocytopenia is one of the hallmarks of the mouseTRALI model²⁰ where it is shown herein that NETs are generated. Reducedplatelet counts have been observed during TRALI in a retrospective studyof patients developing TRALI compared to controls.³⁵ Activation ofplatelets can, in turn, promote additional neutrophil activation andmore NET generation.^(33,36) This has been observed in a sepsis modelwhere LPS was shown to activate platelets through TLR4, promotingplatelet-neutrophil complex formation and NET generation. Also,collagen-mediated platelet activation, such as would occur in trauma ormajor surgery, was linked to platelet-induced NET formation.³³Interestingly, both infection and surgery are risk factors fordeveloping TRALI.^(2,37,38) It is proposed herein that NET formation andits injury to the lung is a common denominator of the differentscenarios causing TRALI.

Indeed, platelets have been shown to contribute to the TRALI mouse modelthat described herein where LPS is given as a primer before antibodyinfusion. Looney and colleagues have shown that after LPS priming,antibody-induced TRALI could be prevented by platelet depletion.²⁰ Arecent study using a model without LPS priming showed that plateletswere not required for TRALI development.²⁷ Even if LPS-activatedplatelets may be involved in vascular cell activation and in NETformation, it was observed herein that platelet depletion withneuraminidase did not prevent NET formation in the lungs of mice withTRALI. However, this result does not exclude that the presence ofplatelets may further augment NET formation.

In most animal models, an antibody infusion is needed or is sufficientto induce TRALI. In both mouse models discussed above, Fcγ receptor(FcγR) activation was implicated in the TRALI process.^(26,27) Sillimanand colleagues have shown in an in vitro study that FcγR are notrequired in neutrophil-mediated damage of endothelial cells at earlytime points after anti-HNA-3a antibody incubation⁵. However, it may notbe the case at later time points where FcγR-binding may lead to NETformation in the lungs during TRALI. The question of whether FcγR areinvolved or not in mouse TRALI was first asked by Looney andcolleagues.²⁶ They have shown that FcγR−/− mice were protected fromTRALI following anti-H-2K^(d) antibody challenge and observed that theinjection of wild-type neutrophils into FcγR−/− mice restored ALI (acutelung injury) following mAb delivery. Strait and collaborators recentlyreported that FcγR were playing a role in TRALI but that they were notresponsible for all the lung injury and that macrophages and complementactivation were also involved.²⁷

It is demonstrated herein that human TRALI-inducing antibodiesstimulated NET generation from primed human neutrophils and thisrequired the Fc portion of the antibody. Just crosslinking of theneutrophil antigen/receptor HNA-3a/CLT-2 was not sufficient to activateNET formation since it was observed herein that anti-HNA-3a F(ab′)₂fragment-treatment on TNF-α-primed neutrophils did not induce NETgeneration. Thus NET initiation by the antibody likely also implicates aFcγR binding-dependent mechanism. Furthermore, when TNF-α-primed humanneutrophils were treated with another antibody that binds neutrophils,more NETs were observed when compared to treatment with an isotypecontrol antibody (data not shown). Thus, described herein is a specificmechanism ultimately leading to NET formation in antibody-mediated TRALIthrough FcγR activation. The data described herein are consistent withstudies describing NET production in autoimmune diseases such assmall-vessel vasculitis¹⁴ or systemic lupus erythematous,^(15,39) whereFcγR can be activated.^(24,40,41)

However, NETs may be formed in non antibody-mediated TRALI as well. Noantibodies could be detected in the transfused units to 2 of the 5 TRALIpatients examined in this study and yet NET biomarkers were generatedand could be detected in their circulation. In 1997, Silliman andcolleagues proposed that biologically active lipids released duringblood storage could activate neutrophils and cause TRALI.^(37,42) Othergroups have also reported effects of components present and accumulatingin the supernatant of stored platelets on neutrophil activation both invitro and in animal models.^(2,22,43-45) A recent study showed thatlonger platelet storage was associated with an increased risk of TRALIin patients.⁴⁶ One such biologically active lipid that could be formedduring platelet storage, platelet-activating factor, is a good inducerof NET formation in vitro²⁹ and other biologically active lipids couldbe as well. Similarly, NETs can form in ALI^(32,47) in which releasedcytokines or bacterial presence have already been described as strongNET inducers.^(6,13,48) Thus it is possible that a variety of stimulantsimplicated in TRALI may alone or in combination with another “hit”induce NET formation.

Lungs may be the most susceptible organ for NET deposition aftertransfusion, as clumped activated neutrophils would likely be trapped inlung microcirculation and transmigrate locally while forming NETs.However, the degradation products of NETs were found systemically inblood of both patients and mice with TRALI. These are likely generatedby DNase 1 which is present in plasma of mice and humans and whosefunction is to degrade DNA released from dying cells.⁴⁹ Indeed, there isno certainty that the increase in NET biomarkers measured in human bloodfrom TRALI patients is due only to the TRALI reaction. That is whyadditional data were provided in order to show that NETs are formed invitro by neutrophils in response to the HNA-3a anti-neutrophil antibodychallenge, and in vivo in the lungs and plasma of originally healthymice suffering from TRALI. As described above herein, supplementing micei.v. with DNase 1 prevented NET-initiated thrombosis in a mouse model ofdeep vein thrombosis⁵⁰ and reduced brain injury in a mouse model ofstroke.⁵¹ In this mouse TRALI model, NETs were revealed in the lungtissue by their irregular patterns when stained for both DNA andcitrullinated histones, a hallmark of NET generation and their DNA basesconfirmed by DNase 1 susceptibility. NETs were found in abundance in theTRALI-affected alveoli with only a few detectable in the pulmonarymicrocirculation. Without wishing to be bound by theory, either they aremore protected there from plasma DNase 1 digestion and therefore areeasily detected or they preferentially form at this location.Monocytes/macrophages were also shown to be important in the developmentof mouse TRALI.²⁷ These cells, in the alveoli, together with lungepithelial cells, generate lung inflammatory mediators such asinterleukin-113 and TNF-α,⁵² both important NET inducers.^(14,17) Thesetwo cytokines could provide a strong local stimulus to the neutrophilsprimed in the vessels by antibodies or bioactive lipids for NETproduction at this location.

Importantly, as described herein, NET deposition in mouse lungs could beinterrupted by DNase 1 inhalation. This treatment not only prevented NETaccumulation in the alveoli but also appeared to improve the lungfunction of the mice experiencing TRALI. Indeed, it seems to be in thealveoli that NETs produced the most preventable damage in TRALI. Inexperiments described herein, i.v. DNase 1 administration did notimprove TRALI outcome, whereas the inhalation of DNase 1 that broughtthe drug directly to the alveoli had a positive effect both whenadministered before or after TRALI onset (FIG. 28A-28C). These resultsare exciting and show that DNase 1 can be used not only as aprophylactic agent but also as a treatment for TRALI after it started.

To conclude, the data described herein document that NETs form duringTRALI both in humans and in mice and that NET degradation, as shownexperimentally by DNase 1 inhalation, can improve the condition of micewith TRALI. Thus a new mechanistic facet in the understanding of thepathogenesis of TRALI is provided and NETs are identified as a targetfor TRALI therapy. DNase 1, the drug we herein, is FDA-approved(PULMOZYME®) to treat the lungs of cystic fibrosis patients. Thesepatients were recently shown to produce NETs markers,⁵³ which were foundin their sputum. It is proposed herein that TRALI-associated NETformation can be arrested by preventing neutrophil chromatindecondensation with neutrophil elastase inhibitors⁵⁴ or with inhibitorsto the histone-citrullinating enzyme peptidylarginine deiminase(PAD4).^(8,55) NET inhibitors can be used to treat TRALI and/or asprophylaxis for patients at high risk of developing TRALI.

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TABLE 2 Description of TRALI patients. These patients' samples wereanalyzed for NET biomarkers as shown in Table 5. PRA: panel reactiveantibody (% of lymphocyte panel reactive with serum); ND: None detected;SOB: Shortness of breath; PRBC: packed red blood cells. TRALI Age/patient # gender Patient leukocyte Abs Donor Abs Clinical Findings 145/F HLA class I 94% PRA Not tested Doveloped chest pain, increased HLAclass II 100% PRA hypotension and SOB 2 h 15 after Nuetrophil Abs oftranfusion with 2 units of PRBCs. unknown specificity. Dense pulmonaryinfiltrates on chest x-ray. Sample was collected 2 h 30 aftertransfusion. 2  7/F HLA class I 25% PRA Non-specific HLA Developed lipswelling, wheezing, Nuetrophil Abs of class I Abs apnea, hypoxia andhypotension unknown specificity 1 h 15 after transfusion with singledonor platelets. Bilateral decreased lung volume, increased opacity inright upper lobe suggestive of atelectasis and consolidation on chestx-ray. Sample ws collected 48 h after transfusion 3 89/F HLA Class Ipositive ND Given 2 units of PRBCs 3 days after open-heart surgery.Developed severe SOB about 3 hours after second unit and was intubatedand given O₂. Striking bilateral pulmonary infiltrates on chest x-ray.Time of sample collection is incertain 4 80/F HLA Class I positive NDAcute SOB and arterial O₂ at 83% HLA Class II positive ND 1 hour afterreceiving 2 units of platelets. Bilateral pulmonary edema on chestx-ray. Time of sample collection is uncertain 5 88/M ND Donor 1 wasmale, Developed acute SOB and arterial never transfused. O₂ at 35% 5 h30 after transfusion of Donor 2 was 2 unitsof PRBCs. Increasedmultiparous female opacities and small pulmonary (4 pregnancies)effusion on chest x-ray. Sample with class I and II was collected 15 hafter transfusion HLA Abs.

TABLE 3 Description of blood donors that induced TRALI reactionsassociated with anti-HNA-3a antibodies. These donors' blood was analyzedin the granulocyte agglutination test and for NET biomarkers in Table 4.INR: international normalized ratio (standardized prothrombin time).Donor Number of HLA Neutrophil number # Age Gender Pregnancies Ab AbRecipient Clinical Findings 1 50 Female 3 Yes HNA-3a A 79-vear old malereceived plasma, including from donor #1. to correct INR before surgicalrepair of pseudoaneurysm. Within 6 hours, developed acute respiratorydistress, hypoxia, and bilateral pulmonary edema found on chest x- ray.2 51 Female 3 No HNA-3a A 67-year old female received 19 blood products,including plasma from donor #2, during surgery to replace mitral andatrial heart valves. Recipient developed severe dyspnea and hypoxia,within 6 hours of blood transfusion. Chest x-ray showed pulmonary edemaand bilateral lung infiltrates and frothy sputum was aspirated fromlungs. Recipient died as a result of the TRALI reaction. 3 65 Female 4No HNA-3a Plasma from donor #3 transfused to male recipient with biliaryobstruction. Recipient, developed chills, rigors, and hypoxia within 6h. Chest x-ray showed pulmonary edema.

TABLE 4 Absence of evidence of circulating NET biomarkers in blooddonors and control individuals. Granulocyte agglutination test (GAT) andNET biomarker analysis in plasma samples from blood donors that inducedTRALI reactions and from control donors. The degree of agglutination wasgraded from − (weak) to 3+ (strong). P = 0.9148, 0.7461 and 0.4921 whenrespectively DNA, MPO and nucleosome levels are compared in plasma fromblood donors that induced TRALI with control individual plasma. Analyseswere performed blinded to sample origin. MPO: myeloperoxidase. Sample #DNA MPO Nucleosomes Specificity GAT (ng/ml) (mU/ml) (OD 405-490) Blooddonor with anti-NB 1 1 +/− 196 6.9 0 Blood donor with anti-HNA-3a 1 3+179 9.9 0.04 Blood donor with anti-HNA-3a 2 2+ 136 0 0 Blood donor withanti-HNA-3a 3 2+ 755 7.1 0 Group median 188 7 0 Control donor 1 − 296 00 Control donor 2 +/− 203 6.3 0 Control donor 3 − 166 1.4 0.02 Controldonor 4 − 173 106.9 0.04 Control donor 5 − 160 8.6 0.03 Control donor 6− 160 0 0.03 Group median 170 4 0 P value 0.9148 0.7461 0.4921

TABLE 5 Evidence of circulating NET biomarkers in serum of patients withTRALI. Granulocyte agglutination test (GAT) and NET biomarker analysisin serum samples from TRALI patients and control healthy subjects. Thedegree of agglutination was graded from − (weak) to 3+ (strong). P =0.0087, 0.1255 and 0.0043 when respectively DNA, MPO and nucleosomelevels are compared in serum from patients with TRALI to control serums.Analysis was performed blinded to sample origin. All the TRALI patients'samples were higher than all control samples for at least one biomarker.MPO: myeloperoxidase. Sample # DNA MPO Nucleosomes Specificity GAT(ng/m1) (mU/m1) (OD 405-490) TRALI patient 1 +/− 479 360.7 0.11 TRALIpatient 2 +/− 473 270.8 0.40 TRALI patient 3 +/− 270 112.4 0.20 TRALIpatient 4 +/− 523 466.5 0.50 TRALI patient 5 +/− 522 55.5 0.11 Groupmedian 479 270.8 0.20 Normal control 1 − 266 39.7 0 Normal control 2 −294 79.6 0.01 Normal control 3 − 250 45.9 0.06 Normal control 4 − 221243 0.10 Normal control 5 − 241 87.4 0.07 Normal control 6 − 235 138.90.05 Group median 245 83.5 0.06 P value 0.0087 0.1255 0.0043

Sequence Listing SEQ ID NO: 1 PAD4 mRNA NCBI Ref Seq: NM_012387    1acagccagag ggacgagcta gcccgacgat ggcccagggg acattgatcc gtgtgacccc   61agagcagccc acccatgccg tgtgtgtgct gggcaccttg actcagcttg acatctgcag  121ctctgcccct gaggactgca cgtccttcag catcaacgcc tccccagggg tggtcgtgga  181tattgcccac ggccctccag ccaagaagaa atccacaggt tcctccacat ggcccctgga  241ccctggggta gaggtgaccc tgacgatgaa agtggccagt ggtagcacag gcgaccagaa  301ggttcagatt tcatactacg gacccaagac tccaccagtc aaagctctac tctacctcac  361cggggtggaa atctccttgt gcgcagacat cacccgcacc ggcaaagtga agccaaccag  421agctgtgaaa gatcagagga cctggacctg gggcccttgt ggacagggtg ccatcctgct  481ggtgaactgt gacagagaca atctcgaatc ttctgccatg gactgcgagg atgatgaagt  541gcttgacagc gaagacctgc aggacatgtc gctgatgacc ctgagcacga agacccccaa  601ggacttcttc acaaaccata cactggtgct ccacgtggcc aggtctgaga tggacaaagt  661gagggtgttt caggccacac ggggcaaact gtcctccaag tgcagcgtag tcttgggtcc  721caagtggccc tctcactacc tgatggtccc cggtggaaag cacaacatgg acttctacgt  781ggaggccctc gctttcccgg acaccgactt cccggggctc attaccctca ccatctccct  841gctggacacg tccaacctgg agctccccga ggctgtggtg ttccaagaca gcgtggtctt  901ccgcgtggcg ccctggatca tgacccccaa cacccagccc ccgcaggagg tgtacgcgtg  961cagtattttt gaaaatgagg acttcctgaa gtcagtgact actctggcca tgaaagccaa 1021gtgcaagctg accatctgcc ctgaggagga gaacatggat gaccagtgga tgcaggatga 1081aatggagatc ggctacatcc aagccccaca caaaacgctg cccgtggtct tcgactctcc 1141aaggaacaga ggcctgaagg agtttcccat caaacgcgtg atgggtccag attttggcta 1201tgtaactcga gggccccaaa cagggggtat cagtggactg gactcctttg ggaacctgga 1261agtgagcccc ccagtcacag tcaggggcaa ggaatacccg ctgggcagga ttctcttcgg 1321ggacagctgt tatcccagca atgacagccg gcagatgcac caggccctgc aggacttcct 1381cagtgcccag caggtgcagg cccctgtgaa gctctattct gactggctgt ccgtgggcca 1441cgtggacgag ttcctgagct ttgtgccagc acccgacagg aagggcttcc ggctgctcct 1501ggccagcccc aggtcctgct acaaactgtt ccaggagcag cagaatgagg gccacgggga 1561ggccctgctg ttcgaaggga tcaagaaaaa aaaacagcag aaaataaaga acattctgtc 1621aaacaagaca ttgagagaac ataattcatt tgtggagaga tgcatcgact ggaaccgcga 1681gctgctgaag cgggagctgg gcctggccga gagtgacatc attgacatcc cgcagctctt 1741caagctcaaa gagttctcta aggcggaagc ttttttcccc aacatggtga acatgctggt 1801gctagggaag cacctgggca tccccaagcc cttcgggccc gtcatcaacg gccgctgctg 1861cctggaggag aaggtgtgtt ccctgctgga gccactgggc ctccagtgca ccttcatcaa 1921cgacttcttc acctaccaca tcaggcatgg ggaggtgcac tgcggcacca acgtgcgcag 1981aaagcccttc tccttcaagt ggtggaacat ggtgccctga gcccatcttc cctggcgtcc 2041tctccctcct ggccagatgt cgctgggtcc tctgcagtgt ggcaagcaag agctcttgtg 2101aatattgtgg ctccctgggg gcggccagcc ctcccagcag tggcttgctt tcttctcctg 2161tgatgtccca gtttcccact ctgaagatcc caacatggtc ctagcactgc acactcagtt 2221ctgctctaag aagctgcaat aaagtttttt taagtcactt tgtacSEQ ID NO: 2 PAD4 amino acid sequence NCBI Ref Seq: NP_036519   1maqgtlirvt peqpthavcv lgtltqldic ssapedctsf sinaspgvvv diahgppakk  61kstgsstwpl dpgvevtltm kvasgstgdg kvqisyygpk tppvkallyl tgveislcad 121itrtgkvkpt ravkdqrtwt wgpcgqgail lvncdrdnle ssamdcedde vldsedlqdm 181slmtlstktp kdfftnhtlv lhvarsemdk vrvfqatrgk lsskcsvvlg pkwpshylmv 241pggkhnmdfy vealafpdtd fpglitltis lldtsnlelp eavvfqdsvv frvapwimtp 301ntqppqevya csifenedfl ksvttlamka kcklticpee enmddqwmqd emeigyiqap 361hktlpvvfds prnrglkefp ikrvmgpdfg yvtrgpqtgg isgldsfgnl evsppvtvrg 421keyplgrilf gdscypsnds rqmhgalqdf lsaqqvgapv klysdwlsvg hvdeflsfvp 481apdrkgfrll lasprscykl fqeqqneghg eallfegikk kkqqkiknil snktlrehns 541fvercidwnr ellkrelgla esdiidipql fklkefskae affpnmvnml vlgkhlgipk 601pfgpvingrc cleekvcsll eplglqctfi ndfftyhirh gevhcgtnvr rkpfsfkwwn 661mvp

What is claimed herein is:
 1. A method of treating or preventingthrombosis in a subject in need thereof, the method comprisingadministering a composition comprising at least one anti-NET compound,wherein the anti-NET compound is a DNase.
 2. The method of claim 1,wherein the thrombosis is caused by leukocyte-derived extracellular DNAtraps.
 3. The method of claim 1, wherein the thrombosis is arterialthrombosis, venous thrombosis, deep vein thrombosis, orinflammation-enhanced thrombosis.
 4. The method of claim 3, wherein thethrombosis is arterial thrombosis.
 5. The method of claim 4, wherein thesubject has thromboembolic occlusion of intracerebral arteries.
 6. Themethod of claim 4, wherein the subject suffers from stroke.
 7. Themethod of claim 1, wherein the thrombosis is associated with acuteinflammation.
 8. The method of claim 1, wherein the composition isadministered by injection or infusion.
 9. The method of claim 8, whereinthe injection is intravenous, intramuscular, subcutaneous, ortransdermal.
 10. The method of claim 1, wherein the infusion isperformed over a 5 minute, 10 minute, 15 minute, 20 minute, or 25 minuteperiod.
 11. The method of claim 10, wherein the administration isrepeated hourly for at least three cycles.
 12. The method of claim 1,wherein the administration is repeated.
 13. The method of claim 12,wherein the administration is repeated daily for at least one week. 14.The method of claim 12, wherein the administration is repeated daily forat least one month.
 15. The method of claim 12, wherein theadministration is repeated weekly.
 16. The method of claim 12, whereinthe administration is repeated biweekly for at least one month.
 17. Themethod of claim 12, wherein the administration is repeated biweekly forat least two months.
 18. The method of claim 12, wherein theadministration is repeated once per month for at least six months. 19.The method of claim 1, wherein the anti-NET compound is administered ata dose of from about 1 μg/kg to about 100 mg/kg
 20. The method of claim19, wherein the anti-NET compound is administered at a dose of from 100μg/kg to 10 mg/kg.
 21. The method of claim 19, wherein the DNase isadministered as a controlled release formulation.
 22. The method ofclaim 19, wherein a single administration of the anti-NET compound tothe subject decreases the concentration of NETs in the patient'sbloodstream by at least 30%.
 23. The method of claim 1, wherein a singleadministration of the anti-NET compound to the subject decreases theconcentration of NETs in the patient's bloodstream by at least 50%. 24.The method of claim 1, wherein the anti-NET compound is administeredprophylactically.
 25. The method of claim 1, wherein the DNase is DNaseI.
 26. The method of claim 1, wherein the subject is furtheradministered at least one anti-thrombotic treatment selected from thegroup consisting of: heparin, tPA, anistreplase, streptokinase,urokinase, a coumadin, warfarin, idraparinux, fondaparinux, aspririn, aadenosine diphosphate receptor inhibitor, a phosphodiesterase inhibitor,a glycoprotein IIB/IIA inhibitor, a adenosine reuptake inhibitor, and athromboxane receptor antagonist.